Helena Carmo

Effect of surface coating on the biodistribution profile of gold nanoparticles in the rat

Successful application of gold nanoparticles (AuNPs) in biomedicine requires extensive safety assessment for which biokinetic studies are crucial. We evaluated the biodistribution of AuNPs (∼20 nm) with different surface coatings: citrate, 11-MUA and 3 pentapeptides, CALNN, CALND and CALNS, after i.v. administration to rats (0.6-1 mg Au/kg). Biodistribution was evaluated based on Au tissue content measured by GFAAS. Citrate-AuNPs were rapidly removed from circulation with 60% of the injected dose depositing in the liver. Thirty minutes post-injection, the lungs presented about 6% of the injected dose with levels decreasing to 0.7% at 24 h. Gold levels in the spleen were of 2.6%. After 24 h, liver presented the highest Au level, followed by spleen and blood. A similar biodistribution profile was observed for MUA-coated AuNPs compared to Cit-AuNPs at 24h post-injection, while significantly higher levels of peptide-capped AuNPs were found in the liver (74-86%) accompanied by a corresponding decrease in blood levels. TEM analysis of liver slices showed AuNPs in Kupffer cells and hepatocytes, trapped inside endosomes. Our data demonstrate that AuNPs are rapidly distributed and that the liver is the preferential accumulation organ. Peptide capping significantly increased hepatic uptake, showing the influence of AuNPs functionalization in biodistribution.

Modulation of P-glycoprotein efflux pump: induction and activation as a therapeutic strategy

P-glycoprotein (P-gp) is an ATP-dependent efflux pump encoded by the MDR1 gene in humans, known to mediate multidrug resistance of neoplastic cells to cancer therapy. For several decades, P-gp inhibition has drawn many significant research efforts in an attempt to overcome this phenomenon. However, P-gp is also constitutively expressed in normal human epithelial tissues and, due to its broad substrate specificity, to its cellular polarized expression in many excretory and barrier tissues, and to its great efflux capacity, it can play a crucial role in limiting the absorption and distribution of harmful xenobiotics, by decreasing their intracellular accumulation. Such a defense mechanism can be of particular relevance at the intestinal level, by significantly reducing the intestinal absorption of the xenobiotic and, consequently, avoiding its access to the target organs. In this review, the current knowledge on this important efflux pump is summarized, and a new focus is brought on the therapeutic interest of inducing and/or activating P-gp for limiting the toxicity caused by its substrates. Several in vivo and in vitro studies validating the use of such a therapeutic strategy are discussed. An extensive literature search for reported P-gp inducers/activators and for the experimental models used in their characterization was conducted. Those studies demonstrate that effective antidotal pathways can be achieved by efficiently promoting the P-gp-mediated efflux of deleterious xenobiotics, resulting in a significant reduction in their intracellular levels and, consequently, in a significant reduction of their toxicity.

Influence of the surface coating on the cytotoxicity, genotoxicity and uptake of gold nanoparticles in human HepG2 cells

The toxicological profile of gold nanoparticles (AuNPs) remains controversial. Significant efforts to develop surface coatings to improve biocompatibility have been carried out. In vivo biodistribution studies have shown that the liver is a target for AuNPs accumulation. Therefore, we investigated the effects induced by ~20 nm spherical AuNPs (0–200 μM Au) with two surface coatings, citrate (Cit) compared with 11‐mercaptoundecanoic acid (11‐MUA), in human liver HepG2 cells. Cytotoxicity was evaluated using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release assays after 24 to 72 h of incubation. DNA damage was assessed by the comet assay, 24 h after incubation with the capped AuNPs. Uptake and subcellular distribution of the tested AuNPs was evaluated by quantifying the gold intracellular content by graphite furnace atomic absorption spectrometry (GFAAS) and transmission electron microscopy (TEM), respectively. The obtained results indicate that both differently coated AuNPs did not induce significant cytotoxicity. An inverse concentration‐dependent increase in comet tail intensity and tail moment was observed in Cit‐AuNPs‐ but not in MUA‐AuNPs‐exposed cells. Both AuNPs were internalized in a concentration‐dependent manner. However, no differences were found in the extent of the internalization between the two types of NPs. Electron‐dense deposits of agglomerates of Cit‐ and MUA‐AuNPs were observed either inside endosomes or in the intercellular spaces. In spite of the absence of cytotoxicity, DNA damage was observed after exposure to the lower concentrations of Cit‐ but not to MUA‐AuNPs. Thus, our data supports the importance of the surface properties to increase the biocompatibility and safety of AuNPs. Copyright

Hepcidin messenger RNA expression in human lymphocytes

Hepcidin regulates intracellular iron levels by interacting with and promoting the degradation of ferroportin, a membrane protein and the only known cellular iron exporter. Studies of hepcidin expression and regulation have focused on its effects in innate immunity and as a regulator of systemic iron metabolism. In the present study we characterized the expression of hepcidin messenger RNA (mRNA) in human peripheral blood mononuclear cells (PBMCs) with a focus on peripheral blood lymphocytes (PBLs). We found that (1) all human PBMCs analyzed express basal hepcidin mRNA levels; (2) hepcidin mRNA expression increases after T‐lymphocyte activation; (3) expression by PBLs increases in response to challenge by holotransferrin (Fe‐TF) and by ferric citrate in vitro; (4) the Fe‐TF‐mediated up‐regulation of hepcidin decreases ferroportin expression at the cytoplasmic membrane of PBLs; and (5) silencing of tumour necrosis factor‐α (TNF‐α) abrogates the effect of Fe‐TF. In summary, we show that hepcidin expression determines intracellular iron levels by regulating the expression of ferroportin, as described in other cells, and that inappropriately low expression of hepcidin impairs normal lymphocyte proliferation. The results establish hepcidin as a new player in lymphocyte biology.

Short- and long-term distribution and toxicity of gold nanoparticles in the rat after a single-dose intravenous administration

UNLABELLED Surface chemistry plays an important role in gold nanoparticles (AuNPs) stability and biocompatibility, which are crucial for their implementation into the clinical setting. We evaluated short- (30 min) and long-term (28 days) biodistribution and toxicity of ~20 nm citrate- and pentapeptide CALNN-coated AuNPs after a single intravenous injection in rats. The pattern of AuNPs distribution in Cit- and CALNN-AuNPs-injected rats was very similar in the assessed time-points. Both AuNPs were quickly removed from the bloodstream and preferentially accumulated in the liver. At 28 days liver remained the main accumulation site but at significantly lower levels compared to those found at 30 min. Spleen atrophy and hematological findings compatible with mild anemia were observed in CALNN-AuNPs-administered rats. Under our experimental conditions, surface coating had more impact on toxicity rather than on biodistribution of the AuNPs. Improvements in the design of capping peptides need to be done to increase biomedical applicability of peptide-coated AuNPs. FROM THE CLINICAL EDITOR The biodistribution and toxicity of ~ 20 nm citrate- and pentapeptide CALNN-coated gold nanoparticles was investigated after a single intravenous injection in rats. Rapid clearance and hepatic accumulation was found at 30-minutes, whereas mild anemia and spleen atrophy was seen 28 days post injection. The authors also concluded that the toxicity was related to the capping proteins as opposed to the biodistribution of the particles, providing important suggestion for future design of gold nanoparticles.

Inhibition of glutathione reductase by isoproterenol oxidation products.

Oxidative stress induced by catecholamines is a well recognized toxic event. This effect has been extensively observed in the heart, where high levels of catecholamines cause enzyme inhibition, lipid peroxidation, energy depletion and myocardial necrosis. Catecholamines can be converted into o-quinones and undergo cyclization into aminochromes. This process can occur enzymatically or through autoxidation and involves the formation of free radicals. Aminochromes are highly reactive molecules that can cause oxidation of protein sulfhydryl groups and deamination catalysis, among other deleterious effects; in addition, inhibition of some enzymes has been also reported. We have studied the effects of isoproterenol oxidation products (IOP) on glutathione reductase (GR) activity in vitro. Isoproterenol (ISO) autoxidation was conducted at 37 degrees C in the dark, for 4 h at pH 7.0 and this process was monitored by UV spectrophotometry at both 340 and 490nm. Addition of the autoxidized solution to GR in the presence of oxidized glutathione (GSSG) and NADPH showed that IOP inhibits GR in a competitive mode and that this effect increases during the 4 h incubation period. This inhibitory effect of IOP was partially prevented by the addition of reduced glutathione (GSH), L-cysteine and ascorbic acid to the reaction mixtures.

Chronic exposure to ethanol exacerbates MDMA-induced hyperthermia and exposes liver to severe MDMA-induced toxicity in CD1 mice

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) is an amphetamine derivative drug with entactogenic, empathogenic and hallucinogenic properties, commonly consumed at rave parties in a polydrug abuse pattern, especially with cannabis, tobacco and ethanol. Since both MDMA and ethanol may cause deleterious effects to the liver, the evaluation of their putative hepatotoxic interaction is of great interest, especially considering that most of the MDMA users are regular ethanol consumers. Thus, the aim of the present study was to evaluate, in vivo, the acute hepatotoxic effects of MDMA (10mg/kg i.p.) in CD-1 mice previously exposed to 12% ethanol as drinking fluid (for 8 weeks). Body temperature was continuously measured for 12h after MDMA administration and, after 24h, hepatic damage was evaluated. The administration of MDMA to non pre-treated mice resulted in sustained hyperthermia, which was significantly increased in ethanol pre-exposed mice. A correspondent higher increase of hepatic heat shock transcription factor (HSF-1) activation was also observed in the latter group. Furthermore, MDMA administration resulted in liver damage as confirmed by histological analysis, slight decrease in liver weight and increased plasma transaminases levels. These hepatotoxic effects were also exacerbated when mice were pre-treated with ethanol. The activities of some antioxidant enzymes (such as SOD, GPx and Catalase) were modified by ethanol, MDMA and their joint action. The hepatotoxicity resulting from the simultaneous exposure to MDMA and ethanol was associated with a higher activation of NF-kappaB, indicating a pro-inflammatory effect in this organ. In conclusion, the obtained results strongly suggest that the consumption of ethanol increases the hyperthermic and hepatotoxic effects associated with MDMA abuse.

Combination effects of amphetamines under hyperthermia - the role played by oxidative stress

Rise in body temperature is a life‐threatening consequence of 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) abuse. We evaluated the impact of hyperthermia on the cytotoxicity of combinations of MDMA and three other amphetamines, often co‐ingested. For this, Hep G2 cells were exposed to MDMA, d‐amphetamine, methamphetamine and 4‐methylthioamphetamine, individually or combined, at 40.5 °C. The results were compared with normothermia data (37.0 °C). Mixture additivity expectations were calculated by independent action and concentration addition (CA) models. To delineate the mechanism(s) underlying the elicited effects, a range of stress endpoints was evaluated, including quantification of reactive oxygen/nitrogen species (ROS/RNS), lipid peroxidation, reduced/oxidized glutathione (GSH/GSSG), ATP and mitochondrial membrane potential (Δψm) changes. Our data show that, in hyperthermia, amphetamines acted additively and mixture effects were accurately predicted by CA. At 40.5 °C, even slight increases in the concentrations of each drug/mixture promoted significant rises in cytotoxicity, which quickly shifted from roughly undetectable to maximal mortality. Additionally, the increase of RNS/ROS production, decrease of GSH, ATP depletion and mitochondrial impairment were exacerbated under hyperthermia. Importantly, when equieffective cytotoxic concentrations of the mixture and individual amphetamines were compared for all tested stress endpoints, mixture effects did not deviate from those elicited by individual treatments, suggesting that these amphetamines have a similar mode of action, which is not altered in combination. Concluding, our data indicate that amphetamine mixtures produce deleterious effects, even when individual drugs are combined at negligible concentrations. These effects are strongly exacerbated in hyperthermia, emphasizing the potential increased risks of ecstasy intake, especially when hyperthermia occurs concurrently with polydrug abuse. Copyright

Copper enhances isoproterenol toxicity in isolated rat cardiomyocytes: effects on oxidative stress.

Sustained high levels of circulating catecholamines may result in cardio-toxicity. Although cardiotoxicity could occur primarily via adrenoceptor activation, there is increasing evidence that it may also occur through oxidative mechanisms. In fact, catecholamines can be converted into aminochromes by auto-oxidation, enzymatically or metal catalyzed, with the concomitant production of reactive intermediates and free radicals. Nevertheless, there is only scarce information concerning the effects of the catecholamine oxidation process on isolated cardiomyocytes.The aim of this work was to evaluate the cardiotoxic effects of isoproterenol (ISO) and its oxidation process in freshly isolated adult rat cardiomyocytes by assessing the cell shape, lactate dehydrogenase leakage, reduced and oxidized glutathione content, and glutathione reductase, peroxidase, and trans-ferase activities. ISO was incubated at concentrations of 0.1, 0.5, and 1 mM in cardiomyocyte suspensions at subphysiological and physiological Ca2+ concentrations for 4h. The same study was repeated in the presence of 20 μM of Cu2+. The levels of ISO in the incubation medium were monitored throughout the assays.Isoproterenol (1 mM) induced both glutathione oxidation and conjugation, but this effect decreased at subphysiological Ca2+ concentrations. The concominant incubation with Cu2+ increased ISO oxidation and increased the glutathione oxidation but decreased the extent of glutathione conjugation. Although only a partial ISO oxidation was observed for all studied ISO concentrations in the presence of copper, the underlying oxidative process or its oxidation products, or both, were sufficient to induce a loss of cardiomyocyte viability and a decrease in the glutathione reductase, peroxidase, and transferase activitles. Thus, the results suggest that the oxidation of catecholamines could be a major mechanism for catecholamine-induced cardiotoxicity.

Synergistic toxicity of ethanol and MDMA towards primary cultured rat hepatocytes.

Ethanol is frequently consumed along with 3,4-methylenedioxymethamphetamine (MDMA; ecstasy). Since both compounds are hepatotoxic and are metabolized in the liver, an increased deleterious interaction resulting from the concomitant use of these two drugs seems plausible. Another important feature of MDMA-induced toxicity is hyperthermia, an effect known to be potentiated after continuous exposure to ethanol. Considering the potential deleterious interaction, the aim of the present study was to evaluate the hepatotoxic effects of ethanol and MDMA mixtures to primary cultured rat hepatocytes and to elucidate the mechanism(s) underlying this interaction. For this purpose, the toxicity induced by MDMA to primary cultured rat hepatocytes in absence or in presence of ethanol was evaluated, under normothermic (36.5 degrees C) and hyperthermic (40.5 degrees C) conditions. While MDMA and ethanol, by themselves, had discrete effects on the analysed parameters, which were slightly aggravated under hyperthermia, the simultaneous incubation of MDMA and ethanol for 24h, resulted in high cell death ratios accompanied by a significant disturbance of cellular redox status and decreased energy levels. Evaluation of apoptotic/necrotic features provided clear evidences that the cell death occurs preferentially through a necrotic pathway. All the evaluated parameters were dramatically aggravated when cells were incubated under hyperthermia. In conclusion, co-exposure of hepatocytes to ethanol and MDMA definitely results in a synergism of the hepatotoxic effects, through a disruption of the cellular redox status and enhanced cell death by a necrotic pathway in a temperature-dependent extent.