Clinical use of bioelectrical impedance analysis in patients affected by myotonic dystrophy type 1: A cross-sectional study.

OBJECTIVES: Myotonic dystrophy type 1 (DM1) is an inherited muscle disorder characterized by slowly progressive weakness due to muscle degeneration. The Muscular Impairment Rating Scale (MIRS) is validated to assess clinical muscle severity of patients with DM1, although the scale is not sensitive enough to assess disease progression in time intervals fit for clinical trials. The aim of this study was to analyze bioelectrical whole body and arm segmental parameters in patients with DM1 to explore a correlation between bioelectrical impedance analysis (BIA) parameters and disease stage. METHODS: Forty patients with DM1 were enrolled in a cross-sectional study. In all patients, MIRS, handgrip strength (HGS), and BIA were assessed. A Kruskal-Wallis test was used to assess the difference in continuous variables according to MIRS. Correlation between BIA values and HGS were made by Pearson's coefficient analysis. A linear regression analysis was performed. RESULTS: Eighteen of 40 patients were men (45%). The median age of the cohort was 42 y (30-58 y). Four patients (10%) were classified as MIRS 1; 20 (50%) MIRS 2; 11 (27.5%) MIRS 3; and 5 (12.5%) as MIRS 4. A correlation was observed between phase angle and MIRS (P = 0.0001). MIRS correlated with other BIA values such as resistance, impedance ratio, and capacitance (P = 0.005, P = 0.0001, P = 0.0006, respectively). At linear regression analysis, segmental resistance, phase angle, impedance ratio, and capacitance of both arms significantly correlated with HGS. CONCLUSIONS: Results from the study support the use of BIA as a suitable procedure for staging DM1 muscle involvement and as a measure of muscle disease outcome, in clinical practice and in clinical trial design of therapeutic drugs.

Chromographic Analysis and Cytotoxic Effects of Chlorhexidine and Sodium Hypochlorite Reaction Mixtures.

INTRODUCTION: The literature reveals controversies regarding the formation of para-chloroaniline (PCA) when chlorhexidine (CHX) is mixed with sodium hypochlorite (NaOCl). This study aimed to investigate the stability of PCA in the presence of NaOCl and to examine the in vitro cytotoxic effects of CHX/NaOCl reaction mixtures. METHODS: Different volumes of NaOCl were added to CHX (mix 1) or PCA (mix 2). Upon centrifugation, the supernatant and precipitate fractions collected from samples were analyzed using high-performance liquid chromatography. The cytotoxic effects of both fractions were examined on human periodontal ligament and 3T3 fibroblast cell lines. RESULTS: High-performance liquid chromatographic analysis showed no PCA signal when NaOCl was mixed with CHX (mix 1). In mix 2, the intensity of PCA was decreased when NaOCl was added to PCA, and chromatographic signals, similar to that of CHX/NaOCl, were also observed. The mortality of precipitates exerted on both cell lines was lower compared with that of supernatants. CONCLUSIONS: The discrepancy in the data from the literature could be caused by the instability of the PCA in the presence of NaOCl. The CHX/NaOCl reaction mixture exhibits a wide range of cytotoxic effects.

Topoisomerases and Anthracyclines: Recent Advances and Perspectives in Anticancer Therapy and Prevention of Cardiotoxicity.

Topoisomerases are ubiquitous enzymes involved in maintaining genomic stability of the cell by regulating the over- or underwinding of DNA strands. Besides their customary functions, topoisomerases are important cellular targets of widely used anticancer drugs. In particular, topoisomerase IIα (Top2α) has been postulated as the primary molecular target of anthracycline's anticancer activity, whereas topoisomerase IIß (Top2ß), the only Top2 present in heart tissue, seems to be involved in the development of anthracycline-induced cardiotoxicity. Noteworthy, cardiotoxicity is the most frequent adverse effect of both conventional and modern anticancer targeted therapy, representing the leading noncancer-related cause of morbidity and mortality in long-term survivors. The molecular mechanisms of anthracyclineinduced cardiotoxicity have been investigated for decades and, despite the numerous mechanistic hypotheses put forward, its aetiology and pathogenesis still remain controversial. This review is aimed at focusing on the double edge sword of topoisomerase-anthracycline interaction, and, in particular, on the potential role of topoisomerases in anthracyclines anticancer activity as well as in the pathogenesis of anthracycline-induced cardiotoxicity.

Cancer Biomarkers Discovery and Validation: State of the Art, Problems and Future Perspectives.

Cancer is one of the major public health problems worldwide representing the leading cause of morbidity and mortality in industrialized countries. To reduce cancer morbidity and mortality as well as to facilitate the evolution from the traditional "one size fits all" strategy to a new "personalized" cancer therapy (i.e., the right drug to the right patient at the right time, using the right dose and schedule), there is an urgent need of reliable, robust, accurate and validated cancer biomarker tests.Unfortunately, despite the impressive advances in tumor biology research as well as in high-powerful "omics" technologies, the translation of candidate cancer biomarkers from bench to bedside is lengthy and challenging and only a few tumor marker tests have been adopted successfully into routine clinical care of oncologic patients.This chapter provides an updated background on biomarkers research in oncology, including biomarkers clinical uses, and discusses the problems of discovery pipeline, biomarkers failures and future perspectives.

Inhibition of Anthracycline Alcohol Metabolite Formation in Human Heart Cytosol: A Potential Role for Several Promising Drugs.

The clinical efficacy of anthracyclines (e.g., doxorubicin and daunorubicin) in cancer therapy is limited by their severe cardiotoxicity, the etiology of which is still not fully understood. The development of anthracycline-induced cardiomyopathy has been found to correlate with myocardial formation and accumulation of anthracycline secondary alcohol metabolites (e.g., doxorubicinol and daunorubicinol) that are produced by distinct cytosolic NADPH-dependent reductases. The aim of the current study is to identify chemical compounds capable of inhibiting myocardial reductases implied in anthracycline reductive metabolism in an attempt to decrease the production of cardiotoxic C-13 alcohol metabolites. Among the variety of tested compounds (metal chelators, radical scavengers, antioxidants, ß-blockers, nitrone spin traps, and lipid-lowering drugs), ebselen, cyclopentenone prostaglandins, nitric oxide donors, and short-chain coenzyme Q analogs resulted in being effective inhibitors of both doxorubicinol and daunorubicinol formation. In particular, ebselen (as well as ebselen diselenide, its storage form in the cells) was the most potent inhibitor of cardiotoxic anthracycline alcohol metabolites with 50% inhibition of doxorubicinol formation at 0.2 mol Eq of ebselen with respect to doxorubicin concentration. The high efficacy, together with its favorable pharmacological profile (low toxicity, lack of adverse effects, and metabolic stability) portends ebselen as a promising cardioprotective agent against anthracycline-induced cardiotoxicity.

Oxidative stress and metabolic syndrome: Effects of a natural antioxidants enriched diet on insulin resistance.

BACKGROUND & AIMS: Oxidative stress (OS) could play a role in metabolic syndrome-related manifestations contributing to insulin resistance (IR). The aim of the present study was to gain insight the relationships between OS, IR and other hormones involved in caloric balance, explaining the effects of a natural antioxidant-enriched diet in patients affected by metabolic syndrome. METHODS: We investigated the effects of dietary antioxidants on IR, studying 53 obese (20 males and 33 females, 18-66 years old, BMI 36.3 ± 5.5 kg/m2), with IR evaluated by Homeostasis Model Assessment (HOMA)-index, comparing 4 treatments: hypocaloric diet alone (group A) or plus metformin 1000 mg/daily (group B), natural antioxidants-enriched hypocaloric diet alone (group C) or plus metformin (group D). A personalized program, with calculated antioxidant intake of 800-1000 mg/daily, from fruit and vegetables, was administered to group C and D. The glycemic and insulinemic response to oral glucose load, and concentrations of total-, LDL- and HDL-cholesterol, triglycerides, uric acid, C reactive protein, fT3, fT4, TSH, insulin-like growth factor 1 were evaluated before and after 3-months. Plasma Total antioxidant capacity was determined by H2O2-metmyoglobin system, which interacting with the chromogen ABTS generates a radical with latency time (LAG) proportional to antioxidant content. RESULTS: Despite a similar BMI decrease, we found a significant decrease of HOMA and insulin peak only in group B and D. Insulin response (AUC) showed the greatest decrease in group D (25.60  ±  8.96%) and was significantly lower in group D vs B. No differences were observed in glucose response, lipid metabolism and TAC (expressed as LAG values). TSH values were significantly suppressed in group D vs B. CONCLUSIONS: These data suggest that dietary antioxidants ameliorate insulin-sensitivity in obese subjects with IR by enhancing the effect of insulin-sensitizing drugs albeit with molecular mechanisms which remain yet to be elucidated.

Effects of dental methacrylates on oxygen consumption and redox status of human pulp cells.

UNLABELLED: Several studies have already demonstrated that the incomplete polymerization of resin-based dental materials causes the release of monomers which might affect cell metabolism. The aim of this study was to investigate the effects of triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, urethane dimethacrylate, and 2-hydroxyethyl methacrylate on (1) cellular energy metabolism, evaluating oxygen consumption rate, glucose consumption, glucose 6-phosphate dehydrogenase activity, and lactate production, and (2) cellular redox status, through the evaluation of glutathione concentration and of the activities of enzymes regulating glutathione metabolism. METHODS: Human pulp cells were used and oxygen consumption was measured by means of a Clark electrode. Moreover, reactive oxygen species production was quantified. Enzymatic activity and glucose and lactate concentrations were determined through a specific kit. RESULTS: Triethylene glycol dimethacrylate, 1,4-butanediol dimethacrylate, and 2-hydroxyethyl methacrylate induced a decrease in oxygen consumption rate, an enhancement of glucose consumption, and lactate production, whilst glucose 6-phosphate dehydrogenase and glutathione reductase activity were not significantly modified. Moreover, the monomers induced an increase of reactive oxygen species production with a consequent increase of superoxide dismutase and catalase enzymatic activities. A depletion of both reduced and total glutathione was also observed. CONCLUSION: The obtained results indicate that dental monomers might alter energy metabolism and glutathione redox balance in human pulp cells.

Anthracyclines and mitochondria.

Anthracyclines remain the cornerstone in the treatment of many malignancies including lymphomas, leukaemias, and sarcomas. Unfortunately, the clinical use of these potent chemotherapeutics is severely limited by the development of a progressive dose-dependent cardiomyopathy that irreversibly evolves toward congestive heart failure. The molecular mechanisms responsible for anthracycline anticancer activity as well as those underlying anthracycline-induced cardiotoxicity are incompletely understood and remain a matter of remarkable controversy. Anthracyclines have long been considered to induce cardiotoxicity by mechanisms different from those mediating their anticancer activity. In particular, anthracycline antitumor efficacy is associated with nuclear DNA intercalation, topoisomerase II inhibition and drug-DNA adducts formation, whereas the cardiotoxicity is prevalently ascribed to oxidative stress and mitochondrial dysfunction. At present, however, the view that distinct mechanisms are implied in anticancer and cardiotoxic responses to anthracycline therapy does not seem fully convincing since beneficial (anticancer) and detrimental (cardiotoxic) effects are to some extent overlapping, share the subcellular organelle targets, the molecular effectors and the pathophysiological processes (i.e. DNA strand breaks, oxidative stress, signalling pathways, mitochondrial dysfunctions, apoptosis etc.).Here, we review the potential role of mitochondria in the molecular mechanisms underlying anthracyclines anticancer activity as well as in the pathogenesis of anthracycline-induced cardiotoxicity.

Mammalian life-span determinant p66shcA mediates obesity-induced insulin resistance.

Obesity and metabolic syndrome result from excess calorie intake and genetic predisposition and are mechanistically linked to type II diabetes and accelerated body aging; abnormal nutrient and insulin signaling participate in this pathologic process, yet the underlying molecular mechanisms are incompletely understood. Mice lacking the p66 kDa isoform of the Shc adaptor molecule live longer and are leaner than wild-type animals, suggesting that this molecule may have a role in metabolic derangement and premature senescence by overnutrition. We found that p66 deficiency exerts a modest but significant protective effect on fat accumulation and premature death in lepOb/Ob mice, an established genetic model of obesity and insulin resistance; strikingly, however, p66 inactivation improved glucose tolerance in these animals, without affecting (hyper)insulinaemia and independent of body weight. Protection from insulin resistance was cell autonomous, because isolated p66KO preadipocytes were relatively resistant to insulin desensitization by free fatty acids in vitro. Biochemical studies revealed that p66shc promotes the signal-inhibitory phosphorylation of the major insulin transducer IRS-1, by bridging IRS-1 and the mTOR effector p70S6 kinase, a molecule previously linked to obesity-induced insulin resistance. Importantly, IRS-1 was strongly up-regulated in the adipose tissue of p66KO lepOb/Ob mice, confirming that effects of p66 on tissue responsiveness to insulin are largely mediated by this molecule. Taken together, these findings identify p66shc as a major mediator of insulin resistance by excess nutrients, and by extension, as a potential molecular target against the spreading epidemic of obesity and type II diabetes.

Mitochondrial damage and metabolic compensatory mechanisms induced by hyperoxia in the U-937 cell line.

Experimental hyperoxia represents a suitable in vitro model to study some pathogenic mechanisms related to oxidative stress. Moreover, it allows the investigation of the molecular pathophysiology underlying oxygen therapy and toxicity. In this study, a modified experimental set up was adopted to accomplish a model of moderate hyperoxia (50% O(2), 96 h culture) to induce oxidative stress in the human leukemia cell line, U-937. Spectrophotometric measurements of mitochondrial respiratory enzyme activities, NMR spectroscopy of culture media, determination of antioxidant enzyme activities, and cell proliferation and differentiation assays were performed. The data showed that moderate hyperoxia in this myeloid cell line causes: i) intriguing alterations in the mitochondrial activities at the levels of succinate dehydrogenase and succinate-cytochrome c reductase; ii) induction of metabolic compensatory adaptations, with significant shift to glycolysis; iii) induction of different antioxidant enzyme activities; iv) significant cell growth inhibition and v) no significant apoptosis. This work will permit better characterization the mitochondrial damage induced by hyperoxia. In particular, the data showed a large increase in the succinate cytochrome c reductase activity, which could be a fundamental pathogenic mechanism at the basis of oxygen toxicity.

Mitochondrial respiratory chain dysfunction, a non-receptor-mediated effect of synthetic PPAR-ligands: biochemical and pharmacological implications.

Peroxisome proliferator activated receptors (PPARs) are a class of nuclear receptors involved in lipid and glucidic metabolism, immune regulation, and cell differentiation. Many of their biological activities have been studied by using selective synthetic activators (mainly fibrates and thiazolidinediones) which have been already employed in therapeutic protocols. Both kinds of drugs, however, showed pharmacotoxicological profiles, which cannot be ascribed by any means to receptor activation. To better understand these non-receptorial or extrareceptorial aspects, the effect of different PPAR-ligands on the metabolic status of human HL-60 cell line has been investigated. At this regard, NMR analysis of cell culture supernatants was accomplished in order to monitor modifications at the level of cell metabolism. Cell growth and chemiluminescence assays were employed to verify cell differentiation. Results showed that all the considered PPAR-ligands, although with different potencies and independently from their PPAR binding specificity, induced a significant derangement of the mitochondrial respiratory chain consisting in a strong inhibition of NADH-cytochrome c reductase activity. This derangement has been shown to be strictly correlated to the adaptive metabolic modifications, as evidenced by the increased formation of lactate and acetate, due to the stimulation of anaerobic glycolysis and fatty acid beta-oxidation. It is worthy noting that the mitochondrial dysfunction appeared also linked to the capacity of any given PPAR-ligand to induce cell differentiation. These data could afford an explanation of biochemical and toxicological aspects related to the therapeutic use of synthetic PPAR-ligands and suggest a revision of PPAR pathophysiologic mechanisms.

Methionine 35 oxidation reduces toxic effects of the amyloid beta-protein fragment (31-35) on human red blood cell.

Amyloid beta-peptide, the central constituent of senile plaques in Alzheimer's disease brain, has been shown to be a source of free radical oxidative stress that may lead to neurodegeneration. In particular, it is well known that oxidation of methionine 35, is strongly related to the pathogenesis of Alzheimer's disease, since it represents the residue in the beta-amyloid peptide most susceptible to oxidation "in vivo". In this study, the fragment 31-35 of the beta-amyloid peptide, which has a single methionine at residue 35, was used to investigate the influence of the oxidation state of methionine-35 on the beta-amyloid peptide (31-35) mediated cytotoxic effects. Because no extensive studies have yet addressed whether amyloid beta peptides-mediated toxic effects can occur in the absence of mitochondria, human red blood cells were used as cell model. Exposure of intact red blood cells to beta-amyloid peptide (31-35) induced a marked stimulation (approximately 45%) of the pentose phosphate pathway and a significant inhibition of the red cell enzyme catalase, compared with the results observed in control red blood cells. In contrast, exposure of red blood cells to the beta-amyloid peptide (31-35)-Met35OX i.e. in which the sulfur of methionine is oxidised to sulfoxide, induced a slight activation of PPP (approximately 19%), and an inhibition of catalase activity lower with respect to the results observed in beta-amyloid peptide (31-35)-treated red blood cells. Since the activities of red cell phosphofructokinase, glucose-6-phosphate dehydrogenase, glutathione peroxidase, glutathione reductase and the functionality of hemoglobin were not modified within the red cell following to beta-amyloid peptides exposure, it is likely that beta-amyloid (31-35)-catalase interaction may represent a selective toxic event. Together, these results support the hypothesis that Abeta peptide and the oxidative state of Met-35 may be involved in the mechanisms responsible of neurodegeneration in Alzheimer's disease.

Anthracycline secondary alcohol metabolite formation in human or rabbit heart: biochemical aspects and pharmacologic implications.

Clinical use of the anticancer anthracyclines doxorubicin (DOX) and daunorubicin (DNR) is limited by development of cardiotoxicity upon chronic administration. Secondary alcohol metabolites, formed after two-equivalent reduction of a carbonyl group in the side chain of DOX or DNR, have been implicated as potential mediators of chronic cardiotoxicity. In the present study we characterized how human heart converted DOX or DNR to their alcohol metabolites DOXol or DNRol. Experiments were carried out using post-mortem myocardial samples obtained by ethically-acceptable procedures, and results showed that DOXol and DNRol were formed by flavin-independent cytoplasmic reductases which shared common features like pH-dependence and requirement for NADPH, but not NADH, as a source of reducing equivalents. However, studies performed with inhibitors exhibiting absolute or mixed specificity toward best known cytoplasmic reductases revealed that DOX and DNR were metabolized to DOXol or DNRol through the action of distinct enzymes. Whereas DOX was converted to DOXol by aldehyde-type reductase(s) belonging to the superfamily of aldo-keto reductases, DNR was converted to DNRol by carbonyl reductase(s) belonging to the superfamily of short-chain dehydrogenase/reductases. This pattern changed in cardiac cytosol derived from rabbit, a laboratory animal often exploited to reproduce cardiotoxicity induced by anthracyclines and to develop protectants for use in cancer patients. In fact, only carbonyl reductases were involved in metabolizing DOX and DNR in rabbit cardiac cytosol, although with different K(m) and V(max). Collectively, these results demonstrate that human myocardium convert DOX and DNR to DOXol or DNRol by virtue of different reductases, an information which may be of value to prevent alcohol metabolite formation during the course of anthracycline-based anticancer therapy. These results also raise caution on the preclinical value of animal models of anthracycline cardiotoxicity, as they demonstrate that the metabolic routes leading to DOXol in a laboratory animal may not be the same as those occurring in patients.

Total antioxidant capacity in patients with varicoceles.

OBJECTIVE: To explore a possible molecular defect linked to infertility, studying total antioxidant capacity (TAC) of seminal plasma in varicocele (VAR). DESIGN: Case-series study. SETTINGS: Volunteers in an academic research environment and out-patients in clinical service. PATIENT(S): Twenty-five VAR patients (9 oligospermic and 16 normospermic) vs 24 non-VAR controls (7 subjects with idiopathic oligospermia and 17 normospermic subjects). INTERVENTION(S): Evaluation of seminal plasma TAC. MAIN OUTCOME MEASURE(S): TAC was measured using myoglobin, as a source of radicals, which interact with a chromogen 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS), whose radical cation is spectroscopically detectable. The latency phase (Lag) in the accumulation of ABTS cation is proportional to antioxidant concentration. RESULT(S): Lag showed significantly greater values in the all VAR patients vs non-VAR subjects. Oligospermic-VAR patients showed the greatest values. Lag and sperm motility significantly correlated in VAR normospermic patients. Follicle-stimulating hormone (FSH) showed significant inverse association with Lag in same group. CONCLUSION(S): The augmented Lag values could indicate an ineffective utilization of antioxidants in oligospermic-VAR, while in normo-VAR the direct correlation between TAC and motility suggest a potential protective role toward sperm motility. In the same group, the inverse correlation with FSH suggests that greater FSH levels induce a better utilization of antioxidants by spermatozoa.