Cellular Toxicity Mechanisms and the Role of Autophagy in Pt(IV) Prodrug-Loaded Ultrasmall Iron Oxide Nanoparticles Used for Enhanced Drug Delivery.

Ultrasmall iron oxide nanoparticles (<10 nm) were loaded with cis-diamminetetrachloroplatinum (IV), a cisplatin (II) prodrug, and used as an efficient nanodelivery system in cell models. To gain further insight into their behavior in ovarian cancer cells, the level of cellular incorporation as well as the platination of mitochondrial and nuclear DNA were measured using inductively coupled plasma mass spectrometry (ICP-MS) strategies. Quantitative Pt results revealed that after 24 h exposure to 20 µM Pt in the form of the Pt(IV)-loaded nanoparticles, approximately 10% of the incorporated Pt was associated with nuclear DNA. This concentration increased up to 60% when cells were left to stand in drug-free media for 3 h. These results indicated that the intracellular reducing conditions permitted the slow release of cisplatin (II) from the cisplatin (IV)-loaded nanoparticles. Similar results were obtained for the platination of mitochondrial DNA, which reached levels up to 17,400 ± 75 ng Pt/ mg DNA when cells were left in drug-free media for 3 h, proving that this organelle was also a target for the action of the released cisplatin (II). The time-dependent formation of Pt-DNA adducts could be correlated with the time-dependent decrease in cell viability. Such a decrease in cell viability was correlated with the induction of apoptosis as the main route of cell death. The formation of autophagosomes, although observed upon exposure in treated cells, does not seem to have played an important role as a means for cells to overcome nanoparticles' toxicity. Thus, the designed nanosystem demonstrated high cellular penetration and the "in situ" production of the intracellularly active cisplatin (II), which is able to induce cell death, in a sustained manner.

Quantitative assessment of the metabolic products of iron oxide nanoparticles to be used as iron supplements in cell cultures.

Iron nanoparticles (NPs) metabolism is directly associated to human health due to their use as anemia treatment and should be studied in detail in cells. Here we present a speciation strategy for the determination of the metabolic products of iron oxide nanoparticles coated by tartaric and adipic acids in enterocytes-like cell models (Caco-2 and HT-29). Such methodology is based on the use of SDS-modified reversed phase high performance liquid chromatography (HPLC) separation using inductively coupled plasma-mass spectrometry (ICP-MS) as Fe selective detector. Post-column isotope dilution analysis is used as quantification tool by adding 57Fe as isotopically enriched standard. To assess the separation capability of the method, two different iron nanostructures: iron sucrose nanoparticles -Venofer®- used as model suspension and iron tartrate/adipate-modified nanoparticles, both of about 4 nm (core size) were evaluated. The two nanostructures were injected into the system showing good peak profiles and quantitative elution recoveries (>80%) in both cases. In addition, both nanoparticulate fractions could be based-line separated from ionic iron species, which needed to be complexed with 1 mM citrate to elute from the column. Exposed cells up to 0.5 mM of iron tartrate/adipate-modified nanoparticles were specifically treated to extract the internalized NPs and the extracts examined using the proposed strategy. The obtained results revealed the presence of three different fractions corresponding to nanoparticle aggregates, dispersed nanoparticles and soluble iron respectively in a single chromatographic run. Quantitative experiments (column recoveries ranging from 60 to 80%) revealed the presence of the majority of the Fe in the nanoparticulated form (>75%) by summing up the dispersed and aggregate particles. Such experiments point out the high uptake and low solubilization rate of the tartrate/adipate NPs making these structures highly suitable as Fe supplements in oral anemia treatments.

Stanozolol Decreases Bone Turnover Markers, Increases Mineralization, and Alters Femoral Geometry in Male Rats.

Stanozonol (ST) is a synthetic derivative of testosterone; it has anabolic/androgenic activity, increasing both the turnover of trabecular bone and the endocortical apposition of bone. The present study aimed to examine the effects of ST on bone status in rats by bone mineral content, markers of formation and resorption, bone density, and structural and microarchitectural parameters. Twenty male Wistar rats were randomly distributed into two experimental groups corresponding to placebo or ST administration, which consisted of weekly intramuscular injections of 10 mg/kg body weight of ST. Plasma parameters were analyzed by immunoassay. Bone mineral content was determined by spectrophotometry. Bone mineral density (BMD) and structural parameters were measured by peripheral quantitative computed tomography, and trabecular and cortical microarchitecture by micro-computed tomography. Plasma Ca, Mg, and alkaline phosphatase were higher, and urinary Ca excretion, corticosterone, and testosterone concentrations lower in the ST group. Femur Ca content was higher and P content was lower in the ST, whereas osteocalcin, aminoterminal propeptides of type I procollagen, and C-terminal telopeptides of type I collagen were lower. Total cross-sectional, trabecular, and cortical/subcortical areas were lower in the ST. No differences were observed on BMD and area parameters of the diaphysis as well as on trabecular and cortical microarchitecture. The use of ST increases bone mineralization, ash percentage, and Ca and Mg content in femur. In spite of an absence of changes in BMD, geometric metaphyseal changes were observed. We conclude that ST alters bone geometry, leads to low bone turnover, and thus may impair bone quality.

Interaction between orexin A and cannabinoid system in the lateral hypothalamus of rats and effects of subchronic intraperitoneal administration of cannabinoid receptor inverse agonist on food intake and the nutritive utilization of protein.

Crosstalk may occur between cannabinoids and other systems controlling appetite, since cannabinoid receptors are present in hypothalamic circuits involved in feeding regulation, and likely to interact with orexin. In this study, an immunohistochemical approach was used to examine the effect of the intracerebroventricular administration of cannabinoid receptor inverse agonist AM 251 on orexin neuropeptide in the hypothalamic system. AM-activated neurons were identified using c-Fos as a marker of neuronal activity. The results obtained show that AM 251 decreases orexin A immunoreactivity, and that it increases c-Fos-immunoreactive neurons within the hypothalamus when compared with the vehicle-injected control group. We also studied the effects of subchronic intraperitoneal administration of AM 251 on food intake, body weight, and protein utilization. The administration of AM 251 at 1, 2, or 5 mg/kg led to a significant reduction in food intake, along with a significant decrease in the digestive utilization of protein in the groups injected with 1 and 2 mg/kg. There was a dose-related slowdown in weight gain, especially at the doses of 2 and 5 mg/kg, during the initial days of the trial. The absence of this effect in the pair-fed group reveals that any impairment to digestibility was the result of administering AM 251. These data support our conclusion that hypothalamic orexigenic neuropeptides are involved in the reduction of appetite and mediated by the cannabinoid receptor inverse agonist. Furthermore, the subchronic administration of AM 251, in addition to its effect on food intake, has significant effects on the digestive utilization of protein.

Peripheral nerve reconstruction with epsilon-caprolactone conduits seeded with vasoactive intestinal peptide gene-transfected mesenchymal stem cells in a rat model.

OBJECTIVE: Attempts have been made to improve nerve conduits in peripheral nerve reconstruction. We investigated the potential therapeutic effect of a vasoactive intestinal peptide (VIP), a neuropeptide with neuroprotective, trophic and developmental regulatory actions, in peripheral nerve regeneration in a severe model of nerve injury that was repaired with nerve conduits. APPROACH: The sciatic nerve of each male Wistar rat was transected unilaterally at 10 mm and then repaired with Dl-lactic-ε-caprolactone conduits. The rats were treated locally with saline, with the VIP, with adipose-derived mesenchymal stem cells (ASCs) or with ASCs that were transduced with the VIP-expressing lentivirus. The rats with the transected nerve, with no repairs, were used as untreated controls. At 12 weeks post-surgery, we assessed their limb function by measuring the ankle stance angle and the percentage of their muscle mass reduction, and we evaluated the histopathology, immunohistochemistry and morphometry of the myelinated fibers. MAIN RESULTS: The rats that received a single injection of VIP-expressing ASCs showed a significant functional recovery in the ankle stance angle (p = 0.049) and a higher number of myelinated fibers in the middle and distal segments of the operated nerve versus the other groups (p = 0.046). SIGNIFICANCE: These results suggest that utilization of a cellular substrate, plus a VIP source, is a promising method for enhancing nerve regeneration using Dl-lactic-ε-caprolactone conduits and that this method represents a potential useful clinical approach to repairing peripheral nerve damage.

Microsystem-assisted synthesis of carbon dots with fluorescent and colorimetric properties for pH detection.

The present paper describes the use of a microfluidic system to synthesize carbon dots (Cdots) and their use as optical pH sensors. The synthesis is based on the thermal decomposition of ascorbic acid in dimethyl sulfoxide. The proposed microsystem is composed of a fluidic and a thermal platform, which enable proper control of synthesis variables. Uniform and monodispersed 3.3 nm-sized Cdots have been synthesized, the optical characterization of which showed their down/upconversion luminescence and colorimetric properties. The obtained Cdots have been used for pH detection with down and upconverison fluorescent properties as excitation sources. The naked eye or a photographic digital camera has also been implemented as detection systems with the hue parameter showing a linear pH range from 3.5 to 10.2. On the other hand, experiments on the cytotoxicity and permeability of the Cdots on human embryonic kidney cells revealed their adsorption on cells without causing any impact on the cellular morphology.

Aggravation by vanadium of magnesium deficiency in STZ-induced diabetic rats.

This study examined changes in the metabolism of magnesium (Mg), and related serum parameters, following treatment with vanadium (V) in streptozotocin-diabetic rats. Over a period of five weeks, four groups were examined: control, diabetic, diabetic-treated with 1 mg V/day or 3 mg V/day. The V was supplied in drinking water as bis(maltolato)oxovanadium(IV). The Mg levels were measured in food, faeces, urine, serum, muscle, kidney, liver, spleen, heart and femur. Albumin, uric acid, urea, total-cholesterol, LDL-cholesterol, triglycerides, aspartate-aminotransferase and alkaline-phosphatase were determined in serum. In the diabetic group, Mg retained and Mg content in serum and femur decreased, while levels of uric acid, urea, total-cholesterol, LDL-cholesterol, triglycerides and alkaline-phosphatase and aspartate-aminotransferase activity increased compared with control rats. In the diabetic group treated with 1 mg V/day, Mg retained, serum levels of Mg, urea and triglycerides, and alkaline-phosphatase activity remained unchanged, while levels of uric acid, total-cholesterol and LDL-cholesterol increased and the Mg content in femur and aspartate-aminotransferase activity decreased compared with the diabetic untreated group. In the diabetic rats treated with 3 mg V/day, food intake and glycaemia were normal. In this group, Mg content in serum, kidney and femur, levels of urea and aspartate-aminotransferase and alkaline-phosphatase activity decreased, whereas LDL-cholesterol increased, uric acid and total-cholesterol levels remained unchanged in comparison with untreated diabetic rats. In conclusion, although treatment with 3 mg V/day normalised the glycaemia, the hypomagnesaemia and tissue depletion of Mg seen in the diabetic rats, caused by the treatment with V, could have partially contributed to the fact that V did not normalise other serum parameters altered by the diabetes.

Absorption, transport and insulin-mimetic properties of bis(maltolato)oxovanadium (IV) in streptozotocin-induced hyperglycemic rats by integrated mass spectrometric techniques.

The use of V(IV) complexes as insulin-enhancing agents has been increasing during the last decade. Among them, 3-hydroxy-2-methyl-4-pyrone and 2-ethyl-3-hydroxy-4-pyrone (maltol and ethyl maltol, respectively) have proven to be especially suitable as ligands for vanadyl ions. In fact, they have passed phase I and phase II clinical trials, respectively. However, the mechanism through which those drugs exert their insulin-mimetic properties is still not fully understood. Thus, the aim of this study is to obtain an integrated picture of the absorption, biodistribution and insulin-mimetic properties of the bis(maltolato)oxovanadium (IV) (BMOV) in streptozotocin-induced hyperglycaemic rats. For this purpose, BMOV hypoglycaemic properties were evaluated by monitoring both the circulating glucose and the glycohemoglobin, biomarkers of diabetes mellitus. In both cases, the results were drug concentration dependent. Using doses of vanadium at 3 mg/day, it was possible to reduce the glycaemia of the diabetic rats to almost control levels. BMOV absorption experiments have been conducted by intestinal perfusion revealing that approximately 35% of V is absorbed by the intestinal cells. Additionally, the transport of the absorbed vanadium (IV) by serum proteins was studied. For this purpose, a speciation strategy using high-performance liquid chromatography (HPLC) for separation and inductively coupled serum mass spectrometry, ICP-MS, for detection has been employed. The obtained HPLC-ICP-MS results, confirmed by MALDI-MS data, showed evidence that V, administered orally, is uniquely bound to transferrin in rat serum.

Determination by high-performance liquid chromatography of phenylbutazone in samples of plasma from fighting bulls.

The purpose of this study was to investigate the possible presence of phenylbutazone in plasma samples from fighting bulls killed in 2nd and 3rd category bullrings in the province of Salamanca (Spain) in 1998, 1999 and 2000. For quantitative and qualitative determination, a high-performance liquid chromatograph was used, equipped with a photodiode-array detector and setting wavelengths at 240, 254 and 284 nm. The mobile phase optimized for the simultaneous detection of dexamethasone, betamethasone, flunixin and phenylbutazone, was 0.01 M acetic acid pH 3 in methanol (35:65 v/v) at a flow rate of 1 ml/min. Plasma samples were deproteinized with 400 microl of acetonitrile and 20 microl of the supernatant were injected directly into the chromatographic system equipped with a Lichrospher 60 RP select B column and guard column. For the quantitative analysis, standard calibration curves were made in a concentration range between 0.25 and 30 microg/ml, using betamethasone as internal standard. The retention time of phenylbutazone was 8.7 +/- 0.2 min and recovery was 83%. The detection and quantification limits were 0.016 and 0.029, respectively for A=240 nm. The study results show that 17 of the 74 samples analyzed in 1998, 18 of those from 1999 and 10 of those from 2000 were positive for phenylbutazone.

Differentiation of dietary regimene of Iberian swine by means of isotopic analysis of carbon and sulphur in hepatic tissue.

The aim of this work was to differentiate the feed received by Iberian swine during fattening (acorns, feed) and their breed (Iberian or White) using analysis of the stable isotopes of carbon (δ(13)C) and sulphur (δ(34)S) in liver tissue samples. The results obtained in the determination of δ(34)S, using a procedure in which organic and inorganic sulphur are converted into BaSO(4) and the procedure that measures δ(34)S in samples of dried ground liver tissue were compared. Joint analysis of carbon (δ(13)C) and sulphur (δ(34)S) permits the differentiation of swine of different breeds receiving different diets (acorns or feed).

[Impact of AIDS on admissions and mortality in an Internal Medicine Department (1990-1995)]. / Impacto del SIDA en los ingresos y en la mortalidad de un Servicio de Medicina Interna (1990-1995).

OBJECTIVE: To know and analyze the main causes of death in an Internal Medicine Department (MI), as well as the impact of AIDS on admissions and mortality rate in such a Department. MATERIALS AND METHODS: A total of 275 patients out of the 35,521 attended patients from 1990 to 1995 had positive serology to HIV (HIV+). A total of 1,793 deaths were recorded, 42 of which were attributed to AIDS. Clinical and epidemiologic parameters were studied; also, management parameters associated with death were investigated (particularly, those caused by this disease). RESULTS: The overall mortality rate was 5.04%, where as the mortality rate among HIV+ in patients was 15.27%; the primary causes of death were cardiovascular, tumoral, and respiratory disease by decreasing frequency. AIDS was the first cause of death in patients aged less than 35 years. As for AIDS, the number of admissions on account of this disease had a progressive increase along the study period; the mean age of dead patients (31.6 years) tended to increase in the last few years and to be significantly higher among men (6 years); the mean of hospital stay among HIV+ patients (14.16 days) tended to decrease in the last few years. CONCLUSIONS: AIDS has had a progressive increase and has become the primary cause of hospital death in IM, in patients aged less than 35 years. As the experience with this disease increases, the mean stay of HIV+ patients decreases, although it tends to increase in patients who ultimately die, possibly because of the social changes in the last few years, as disease, and particularly death, is intended to become separated from family home.