Cellular uptake and toxicity of gold nanoparticles on two distinct hepatic cell models.

Gold nanoparticles (AuNPs) have huge potential for various biomedical applications, but their successful use depends on their uptake and possible toxicity in the liver, their main site for accumulation. Therefore, in this work we compared the cytotoxic effects induced by AuNPs with different size (~ 15 nm and 60 nm), shape (nanospheres and nanostars) and capping [citrate- or 11-mercaptoundecanoic acid (MUA)], in human HepaRG cells or primary rat hepatocytes (PRH) cultivated with serum-free or Foetal Bovine Serum (FBS)-supplemented media. The safety assessment of the AuNPs demonstrated that overall they present low toxicity towards hepatic cells. Among all the tested AuNPs, the smaller 15 nm spheres displayed the highest toxicity. The toxicological effect was capping, size and cell-type dependent with citrate-capping more toxic than MUA (PRH with FBS), the 15 nm AuNPs more toxic than 60 nm counterparts and PRH more sensitive, as compared to the HepaRG cells. The incubation with FBS-free media produced aggregation of AuNPs while its presence greatly influenced the toxicity outcomes. The cellular uptake of AuNPs was shape, size and capping dependent in PRH cultivated in FBS-supplemented media, and significantly different between the two types of cells with extensively higher internalization of AuNPs in PRH, as compared to the HepaRG cells. These data show that the physical-chemical properties of AuNPs, including size and shape, as well as the type of cellular model, greatly influence the interaction of the AuNPs with the biological environment and consequently, their toxicological effects.

Study of the intestinal uptake and permeability of gold nanoparticles using both in vitro and in vivo approaches.

Gold nanoparticles (AuNPs) are highly attractive to biomedical applications. Here, we investigated the effects of (i) ca. 15 nm spherical AuNPs capped with citrate or 11-mercaptoundecanoic acid (MUA) and (ii) ca. 60 nm spherical citrate-capped AuNPs, and ca. 60 nm MUA-capped star-shaped AuNPs on the cytotoxicity, cellular uptake and permeability, using media supplemented or not with 1% fetal bovine serum (FBS) on caucasian colon adenocarcinoma Caco-2 cells. In addition, the colloidal stability of the nanoparticles in media (supplemented or not) was assessed after 24 h-incubations at 60 µM. The 60 nm gold nanospheres and stars were administrated orally to Wistar rats in order to evaluate their systemic absorption and biodistribution after 24 h. At non-supplemented media settings, citrate-capped gold nanoparticles seem to be more toxic than their MUA-capped counterparts. Also, smaller nanoparticles show higher toxicity than larger ones. The use of cell culture media with 1% FBS not only increased the stability of all AuNPs, as also significantly reduced their cytotoxicity. In the uptake studies, higher AuNPs incorporation was noticed in serum supplemented media, this effect being particularly significant for the 60 nm nanoparticles. Cellular incorporation depended also on the capping agent and size. None of the tested samples crossed the in vitro intestinal barrier. Confirming the in vitro results, the in vivo biodistribution study of the 60 nm AuNPs orally given to rats showed that their systemic absorption is low and that they are mainly eliminated through the faeces. Altogether, these preliminary results suggest that our novel AuNPs have high potential to be considered promising candidates for application in diagnostics or drug delivery at the intestinal level, showing high biocompatibility. However, unless it is desired that these nanomaterials avoid systemic absorption upon oral administration, additional functionalization should be sought to increase their low bioavailability.

A Metabolomic Approach for the In Vivo Study of Gold Nanospheres and Nanostars after a Single-Dose Intravenous Administration to Wistar Rats.

Gold nanoparticles (AuNPs) are promising nanoplatforms for drug therapy, diagnostic and imaging. However, biological comparison studies for different types of AuNPs fail in consistency due to the lack of sensitive methods to detect subtle differences in the expression of toxicity. Therefore, innovative and sensitive approaches such as metabolomics are much needed to discriminate toxicity, specially at low doses. The current work aims to compare the in vivo toxicological effects of gold nanospheres versus gold nanostars (of similar ~40 nm diameter and coated with 11-mercaptoundecanoic acid) 24 h after an intravenous administration of a single dose (1.33 × 1011 AuNPs/kg) to Wistar rats. The biodistribution of both types of AuNPs was determined by graphite furnace atomic absorption spectroscopy. The metabolic effects of the AuNPs on their main target organ, the liver, were analyzed using a GC-MS-based metabolomic approach. Conventional toxicological endpoints, including the levels of ATP and reduced and oxidized glutathione, were also investigated. The results show that AuNPs preferentially accumulate in the liver and, to a lesser extent, in the spleen and lungs. In other organs (kidney, heart, brain), Au content was below the limit of quantification. Reduced glutathione levels increased for both nanospheres and nanostars in the liver, but ATP levels were unaltered. Multivariate analysis showed a good discrimination between the two types of AuNPs (sphere- versus star-shaped nanoparticles) and compared to control group. The metabolic pathways involved in the discrimination were associated with the metabolism of fatty acids, pyrimidine and purine, arachidonic acid, biotin, glycine and synthesis of amino acids. In conclusion, the biodistribution, toxicological, and metabolic profiles of gold nanospheres and gold nanostars were described. Metabolomics proved to be a very useful tool for the comparative study of different types of AuNPs and raised awareness about the pathways associated to their distinct biological effects.

A multiparametric study of gold nanoparticles cytotoxicity, internalization and permeability using an in vitro model of blood-brain barrier. Influence of size, shape and capping agent.

Gold nanoparticles (AuNPs) have biomedical application on imaging and due to increased optical performance, star-shaped AuNPs are of special interest. Because shape, size and capping greatly influence their toxicokinetics and toxicodynamics, a systematic multiparametric comparative study of the influence of these parameters on the cytotoxicity, internalization, and in vitro permeability was conducted in human Cerebral Microvascular Endothelial Cell line (hCMEC/D3), an in vitro model of the human blood-brain barrier (BBB). AuNPs of different size (14 nm and ∼50 nm), shape (spheres and stars), and coating (11-mercaptoundecanoic acid or MUA and sodium citrate) were synthesized and fully characterized. The time- and concentration-dependent cytotoxic profile of the tested AuNPs differed for the different AuNPs. Generally, toxicity was greater for stars relative to sphere-shaped AuNPs, and citrate coating was more toxic than MUA. Regarding the influence of size, smaller-sized AuNPs were more cytotoxic when compared at the same Au concentration. However, when the concentration of AuNPs was expressed as the number of AuNPs/mL, a higher degree of cytotoxicity was noted for the larger ̴50 nm AuNPs. To understand the influence of size, shape and capping, a systematic study design, in which only one of the variables changes, is determinant for correct data interpretation. Considering the results herein presented, for the sake of comparison of differently-sized AuNPs, it is preferable to design the study based upon the number of nanoparticles, since at a fixed Au concentration the number of particles available to promote effect is higher for smaller-sized AuNPs. Cellular internalization also differed among the tested AuNPs; although all were unable to cross the in vitro BBB, the intracellularly accumulated AuNPs can induce cell damage and later compromise BBB integrity and permeability.

Inorganic mercury intoxication: A case report.

Mercury is a heavy metal with unique physico-chemical properties, and it is well distributed throughout the environment, being present in soil, water and air. This non-essential element is considered by the World Health Organization (WHO) as one of the ten most troublesome chemical to public health. Its toxicity spectrum depends on the chemical form in which it presents: elemental (metallic), organic or inorganic. The known intoxications are mainly occupational (mining, agriculture, incineration) or related to the use of dental amalgams or the consumption of contaminated fish and shellfish. Nowadays, acute exposures to toxic amounts of mercury are increasingly rare, especially those involving inorganic mercury compounds. The rate is even lower if we refer to intentional poisonings. Although there is a growing understanding of the toxicokinetics of mercury, there is still a lack of studies that support the emerging theories about its bioavailability in humans. In this manuscript we describe a rare case of an individual who committed suicide by ingesting mercuric oxide. The aim is to offer a medical contribution to the better understanding of the kinetics of this metal, making a discussion based on published literature and analyzing its distribution, metabolism, internal doses, target and reservoir organs. The whole case - clinical course of the victim and her fatal destiny, the ante- and post-mortem sample concentrations and the necropsy findings - illustrates a situation that meets specific features of acute poisoning by ingestion of inorganic mercury, thus constituting an important support towards a more realistic and a based on evidence understanding of mercury biodistribution in humans.

Chromium speciation and biochemical changes vary in relation to plant ploidy.

Uptake of trivalent chromium (Cr(III)-chloride), Cr speciation and consequences for the metabolism in chamomile plants with two ploidy levels have been studied. Depletion of fresh biomass, tissue water content and soluble proteins in response to high (120 µM) Cr(III) was ploidy-independent. Cr mainly accumulated in the roots (only negligibly in the shoots) and total root Cr amount was higher in tetraploid ones including the proof with specific fluorescent indicator (naphthalimide-rhodamine) of Cr(III). Quantification of Cr(VI) detected its higher content in tetraploid roots (up to 4.2% from total Cr), indicating partial oxidation of applied Cr(III). Higher H2O2 presence but lower activities of peroxidases were observed in tetraploid roots while nitric oxide, superoxide dismutase and glutathione reductase activities did not differ extensively. Soluble phenols, lignin, non-protein thiols, individual thiols (glutathione and phytochelatin 2) and ascorbic acid responded to high Cr(III) similarly in both cultivars while decrease of minerals was more pronounced in tetraploid ones. It seems that Cr(III)-induced oxidative stress arises from high root Cr uptake and Cr(VI) presence and is related to depletion of thiols. Assay of Krebs cycle acids confirmed rather depletion under 120 µM Cr(III) in both cultivars but increase in citric acid may indicate its involvement in root Cr chelation. Subsequent comparison of Cr(III)-chloride and Cr(III)-nitrate showed similar influence on Cr accumulation and majority of biochemical responses while different impact on phytochelatin 2 amount was the most distinct feature.

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

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.

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.

Tolerance and bioaccumulation of copper by the entomopathogen Beauveria bassiana (Bals.-Criv.) Vuill. exposed to various copper-based fungicides.

This work evaluates for the first time the relationships between copper-tolerance, -solubilization and -bioaccumulation in the entomopathogen Beauveria bassiana exposed to Bordeaux mixture, copper oxychloride or copper hydroxide. Bordeaux mixture was highly detrimental to fungus, by inhibiting the growth totally at the recommended dose (RD) and 2×RD. Copper hydroxide and copper oxychloride were found to be less toxic, reducing fungus growth, sporulation and conidial germination in an average of 29  %, 30 % and 58 %, respectively. These two copper forms were the easiest to solubilize, to precipitate and the most accumulated by B. bassiana, suggesting the involvement of all these processes on fungus copper-tolerance.

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.

Chromium speciation analysis in bread samples.

Chromium is a controversial element with important essentiality and toxicity, depending on its different species; its speciation analysis in principal human foodstuffs, as in the case of bread, is of utmost importance. With this purpose, a method was validated, including a wet acid digestion procedure for total chromium dissolution, a selective alkaline extraction of hexavalent chromium, and ETAAS determination. The method was applied to the determination of total and hexavalent chromium in 152 bread samples. The total chromium contents were 47.3 +/- 20.0 and 50.9 +/- 22.2 microg/kg of dry weight for white and whole bread samples, respectively; those for hexavalent chromium were 5.65 +/- 5.44 and 6.82 +/- 4.88 microg/kg of dry weight. On the basis of a mean daily ingestion of three bread units, the calculated daily intake was up to 12.7 microg/day for total chromium and 1.98 microg/day for hexavalent chromium. Referring to total chromium, bread can contribute up to 10% of the Reference Daily Intake, 120 microg/day.

Mercury fatal intoxication: two case reports.

We report two cases of fatal intoxications with mercury, one intentional and the other allegedly resulting from a drug formulation mistake. Both cases occurred in the year of 2004. The first case refers to a man who ingested a great portion of a mercuric chloride solution. He attended a hospital emergency, submitted to treatment, but died after 49 days. In the second case, a woman applied on the chest skin an ointment containing a great quantity of mercury bromide. After 7 days of treatment in the hospital, she died. In both cases, samples of tissues and organs were collected at autopsy for mercury analysis. Because methylation of mercury in humans after exposure to metallic or inorganic mercury is almost unknown, both total mercury and methylmercury were quantified in the post-mortem samples. The quantifications were carried out by Cold Vapour Generation Atomic Absorption Spectrometry for total mercury and by HPLC-UV for methylmercury. The total mercury contents found in the post-mortem fluid and tissue samples were consentaneous with mercury poisoning. For the first case, the concentrations found, expressed in microg/g wet weight, were in the liver 49.9, lung 3.27 and brain 0.33, and for blood 11.7 microg/mL. For the second case, the concentrations expressed in microg/g wet weight were in the liver 46.6, lung 14.6, brain 0.21, kidney 77.7, stomach 7.12, spleen 6.4 and heart 2.34, and for blood and urine 2.95 and 1.40 microg/mL, respectively. Only in the first case was methylmercury found and quantified in liver (1.70 microg/g wet weight) and in blood (0.15 microg/mL) samples.

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.

Validation of an electrothermal atomization atomic absorption spectrometry method for the determination of aluminum, copper, and lead in grapes.

An electrothermal atomization atomic absorption spectrometry method was validated to quantify aluminum, copper, and lead in grapes. The limits of detection were 1.11, 0.19, and 0.35 micro g/L for Al, Cu, and Pb, respectively. The linearity ranges under optimized conditions were 1.11-50.0, 0.19-25, and 0.35-50.0 micro g/L for Al, Cu, and Pb, respectively. The limits of quantification were 74.0, 12.5, and 11.6 ng/g of dry weight for Al, Cu, and Pb, respectively. For all of the metals, the precision for the instrumental method was lower than 5.4% and for the analytical method, lower than 10%. The accuracy of the method was evaluated by the standard additions method, the recoveries being higher than 90% for all of the concentrations added. An interference study was also carried out in a simulated matrix, and it was verified that the deviations from the expected values were lower than 3.4% for all of the metals. The method was applied to the monitoring of the metals referred to above in 35 samples of grapes obtained in marketplaces and at farmhouses. The metals were quantified in the whole grapes, washed or not, and in the peel and pulp of unwashed grapes.

Validation of a method to quantify copper and other metals in olive fruit by ETAAS. Application to the residual metal control after olive tree treatments with different copper formulations.

An electrothermal atomization atomic absorption spectrometry method was validated to quantify residues of copper, aluminum, cadmium, chromium, iron, lead, and nickel in olive fruit. The linearity ranges under the optimized conditions were 0.19-20.0, 1.11-50.0, 0.02-2.0, 0.15-20.0, 0.80-20.0, 0.35-50.0, and 0.60-50.0 microg/L, respectively. The limits of quantification were, expressed in nanograms per gram of dry weight, 12.6, 74.0, 1.34, 10.0, 53.4, 23.4, and 40.0, respectively. For all of the metals the precision of the instrumental method was <6.3% and that of the analytical method was always <10%, except for aluminum, for which the precision was 12%. The accuracy of the method was evaluated according to the standard additions method, the recoveries being >90% for all of the added concentrations. An interference study was also carried out in a simulated matrix, and it was verified that the deviations of the expected values were <6% for all of the metals. The method was applied to the monitoring of the residues of the referred metals in olive fruits collected from trees pulverized with three different copper formulations available on the market to control fungal diseases.

Silicon and iron levels in tissues of animals treated with a "ferrimagnetic ceramic" with oncotherapeutic potential (anti-tumor) value.

The stability in a biological environment of an injectable cement with oncotherapeutic potential--consisting of a glass powder of SiO2 (35.6%), CaO (42.4%), P2O5 (17%), Na2O (5%) and 30% of its weight of Fe3O4 dissolved in (NH4)2HPO4 plus NH4H2PO4--was evaluated referring to the release of silicon and iron. The experimental model was the rat, and organs (liver, kidney, spleen, lung, heart, and brain) of the implanted and control animals were collected for quantification of these elements by electrothermal atomization atomic absorption spectrometry methods. In most of the analysed organs no significant difference in the contents of silicon and iron between the implanted and the control animals was found.