Niels Hadrup

Distribution of silver in rats following 28 days of repeated oral exposure to silver nanoparticles or silver acetate

BackgroundThe study investigated the distribution of silver after 28 days repeated oral administration of silver nanoparticles (AgNPs) and silver acetate (AgAc) to rats. Oral administration is a relevant route of exposure because of the use of silver nanoparticles in products related to food and food contact materials.ResultsAgNPs were synthesized with a size distribution of 14 ± 4 nm in diameter (90% of the nanoparticle volume) and stabilized in aqueous suspension by the polymer polyvinylpyrrolidone (PVP). The AgNPs remained stable throughout the duration of the 28-day oral toxicity study in rats. The organ distribution pattern of silver following administration of AgNPs and AgAc was similar. However the absolute silver concentrations in tissues were lower following oral exposure to AgNPs. This was in agreement with an indication of a higher fecal excretion following administration of AgNPs. Besides the intestinal system, the largest silver concentrations were detected in the liver and kidneys. Silver was also found in the lungs and brain. Autometallographic (AMG) staining revealed a similar cellular localization of silver in ileum, liver, and kidney tissue in rats exposed to AgNPs or AgAc.Using transmission electron microscopy (TEM), nanosized granules were detected in the ileum of animals exposed to AgNPs or AgAc and were mainly located in the basal lamina of the ileal epithelium and in lysosomes of macrophages within the lamina propria. Using energy dispersive x-ray spectroscopy it was shown that the granules in lysosomes consisted of silver, selenium, and sulfur for both AgNP and AgAc exposed rats. The diameter of the deposited granules was in the same size range as that of the administered AgNPs. No silver granules were detected by TEM in the liver.ConclusionsThe results of the present study demonstrate that the organ distribution of silver was similar when AgNPs or AgAc were administered orally to rats. The presence of silver granules containing selenium and sulfur in the intestinal wall of rats exposed to either of the silver forms suggests a common mechanism of their formation. Additional studies however, are needed to gain further insight into the underlying mechanisms of the granule formation, and to clarify whether AgNPs dissolve in the gastrointestinal system and/or become absorbed and translocate as intact nanoparticles to organs and tissues.

Oral toxicity of silver ions, silver nanoparticles and colloidal silver - A review

Orally administered silver has been described to be absorbed in a range of 0.4-18% in mammals with a human value of 18%. Based on findings in animals, silver seems to be distributed to all of the organs investigated, with the highest levels being observed in the intestine and stomach. In the skin, silver induces a blue-grey discoloration termed argyria. Excretion occurs via the bile and urine. The following dose-dependent animal toxicity findings have been reported: death, weight loss, hypoactivity, altered neurotransmitter levels, altered liver enzymes, altered blood values, enlarged hearts and immunological effects. Substantial evidence exists suggesting that the effects induced by particulate silver are mediated via silver ions that are released from the particle surface. With the current data regarding toxicity and average human dietary exposure, a Margin of Safety calculation indicates at least a factor of five before a level of concern to the general population is reached.

Quantitative Characterization of Gold Nanoparticles by Field-Flow Fractionation Coupled Online with Light Scattering Detection and Inductively Coupled Plasma Mass Spectrometry

An analytical platform coupling asymmetric flow field-flow fractionation (AF(4)) with multiangle light scattering (MALS), dynamic light scattering (DLS), and inductively coupled plasma mass spectrometry (ICPMS) was established and used for separation and quantitative determination of size and mass concentration of nanoparticles (NPs) in aqueous suspension. Mixtures of three polystyrene (PS) NPs between 20 and 100 nm in diameter and mixtures of three gold (Au) NPs between 10 and 60 nm in diameter were separated by AF(4). The geometric diameters of the separated PS NPs and the hydrodynamic diameters of the Au and PS NPs were determined online by MALS and DLS, respectively. The three separated Au NPs were quantified by ICPMS and recovered at 50-95% of the injected masses, which ranged between approximately 8-80 ng of each nanoparticle size. Au NPs adhering to the membrane in the separation channel was found to be a major cause for incomplete recoveries. The lower limit of detection (LOD) ranged between 0.02 ng Au and 0.4 ng Au, with increasing LOD by increasing nanoparticle diameter. The analytical platform was applied to characterization of Au NPs in livers of rats, which were dosed with 10 nm, 60 nm, or a mixture of 10 and 60 nm nanoparticles by intravenous injection. The homogenized livers were solubilized in tetramethylammonium hydroxide (TMAH), and the recovery of Au NPs from the livers amounted to 86-123% of their total Au content. In spite of successful stabilization with bovine serum albumin even in alkaline medium, separation of the Au NPs by AF(4) was not possible due to association with undissolved remains of the alkali-treated liver tissues as demonstrated by electron microscopy images.

The similar neurotoxic effects of nanoparticulate and ionic silver in vivo and in vitro

We compared the neurotoxic effects of 14 nm silver nanoparticles (AgNPs) and ionic silver, in the form of silver acetate (AgAc), in vivo and in vitro. In female rats, we found that AgNPs (4.5 and 9 mg AgNP/kg bw/day) and ionic silver (9 mg Ag/kg bw/day) increased the dopamine concentration in the brain following 28 days of oral administration. The concentration of 5-hydroxytryptamine (5-HT) in the brain was increased only by AgNP at a dose of 9 mg Ag/kg bw/day. Only AgAc (9 mg Ag/kg bw/day) was found to increase noradrenaline concentration in the brain. In contrast to the results obtained from a 28-day exposure, the dopamine concentration in the brain was decreased by AgNPs (2.25 and 4.5mg/kg bw/day) following a 14-day exposure. These data suggest that there are differential effects of silver on dopamine depending on the length of exposure. In vitro, AgNPs, AgAc and a 12 kDa filtered sub-nano AgNP fraction were used to investigate cell death mechanisms in neuronal-like PC12 cells. AgNPs and the 12 kDa filtered fraction decreased cell viability to a similar extent, whereas AgAc was relatively more potent. AgNPs did not induce necrosis. However, apoptosis was found to be equally increased in cells exposed to AgNPs and the 12kDa filtered fraction, with AgAc showing a greater potency. Both the mitochondrial and the death receptor pathways were found to be involved in AgNP- and AgAc-induced apoptosis. In conclusion, 14 nm AgNPs and AgAc affected brain neurotransmitter concentrations. AgNP affected 5-HT, AgAc affected noradrenaline, whereas both silver formulations affected dopamine. Furthermore, apoptosis was observed in neuronal-like cells exposed to AgNPs, a 12 kDa filtered fraction of AgNP, and AgAc. These findings suggest that ionic silver and a 14 nm AgNP preparation have similar neurotoxic effects; a possible explanation for this could be the release and action of ionic silver from the surface of AgNPs.

Fluorochemicals used in food packaging inhibit male sex hormone synthesis

Polyfluoroalkyl phosphate surfactants (PAPS) are widely used in food contact materials (FCMs) of paper and board and have recently been detected in 57% of investigated materials. Human exposure occurs as PAPS have been measured in blood; however knowledge is lacking on the toxicology of PAPS. The aim of this study was to elucidate the effects of six fluorochemicals on sex hormone synthesis and androgen receptor (AR) activation in vitro. Four PAPS and two metabolites, perfluorooctanoic acid (PFOA) and 8:2 fluorotelomer alcohol (8:2 FTOH) were tested. Hormone profiles, including eight steroid hormones, generally showed that 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH led to decreases in androgens (testosterone, dehydroepiandrosterone, and androstenedione) in the H295R steroidogenesis assay. Decreases were observed for progesterone and 17-OH-progesterone as well. These observations indicated that a step prior to progestagen and androgen synthesis had been affected. Gene expression analysis of StAR, Bzrp, CYP11A, CYP17, CYP21 and CYP19 mRNA showed a decrease in Bzrp mRNA levels for 8:2 monoPAPS and 8:2 FTOH indicating interference with cholesterol transport to the inner mitochondria. Cortisol, estrone and 17β-estradiol levels were in several cases increased with exposure. In accordance with these data CYP19 gene expression increased with 8:2 diPAPS, 8:2 monoPAPS and 8:2 FTOH exposures indicating that this is a contributing factor to the decreased androgen and the increased estrogen levels. Overall, these results demonstrate that fluorochemicals present in food packaging materials and their metabolites can affect steroidogenesis through decreased Bzrp and increased CYP19 gene expression leading to lower androgen and higher estrogen levels.

Concentration Addition, Independent Action and Generalized Concentration Addition Models for Mixture Effect Prediction of Sex Hormone Synthesis In Vitro

Humans are concomitantly exposed to numerous chemicals. An infinite number of combinations and doses thereof can be imagined. For toxicological risk assessment the mathematical prediction of mixture effects, using knowledge on single chemicals, is therefore desirable. We investigated pros and cons of the concentration addition (CA), independent action (IA) and generalized concentration addition (GCA) models. First we measured effects of single chemicals and mixtures thereof on steroid synthesis in H295R cells. Then single chemical data were applied to the models; predictions of mixture effects were calculated and compared to the experimental mixture data. Mixture 1 contained environmental chemicals adjusted in ratio according to human exposure levels. Mixture 2 was a potency adjusted mixture containing five pesticides. Prediction of testosterone effects coincided with the experimental Mixture 1 data. In contrast, antagonism was observed for effects of Mixture 2 on this hormone. The mixtures contained chemicals exerting only limited maximal effects. This hampered prediction by the CA and IA models, whereas the GCA model could be used to predict a full dose response curve. Regarding effects on progesterone and estradiol, some chemicals were having stimulatory effects whereas others had inhibitory effects. The three models were not applicable in this situation and no predictions could be performed. Finally, the expected contributions of single chemicals to the mixture effects were calculated. Prochloraz was the predominant but not sole driver of the mixtures, suggesting that one chemical alone was not responsible for the mixture effects. In conclusion, the GCA model seemed to be superior to the CA and IA models for the prediction of testosterone effects. A situation with chemicals exerting opposing effects, for which the models could not be applied, was identified. In addition, the data indicate that in non-potency adjusted mixtures the effects cannot always be accounted for by single chemicals.

Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting.

The mechanisms underlying targeted sorting of endocytosed receptors for recycling to the plasma membrane or degradation in lysosomes are poorly understood. In this report, the C‐terminal tails of the five dopamine receptors (D1–D5) were expressed as glutathione S‐transferase (GST) fusion proteins and studied for their interaction with ezrin–radixin–moesin‐binding phosphoprotein 50 (EBP50) and N‐ethylmaleimide‐sensitive factor (NSF), which are known to be involved in post‐endocytic recycling of receptors back to the plasma membrane, and with sorting nexin 1 (SNX1), known to be involved in targeting receptors to lysosomal degradation. EBP50 did not bind any of the dopamine receptor tails. NSF bound strongly to D1 and D5 and only weakly to D2, D3 and D4. However, SNX1 clearly distinguished between D1 and D5, as only D5 bound strongly to this protein. This report shows that there are distinct interaction patterns for NSF and SNX1 to the various dopamine receptor subtypes.

Selection of reference genes for quantitative RT-PCR (RT-qPCR) analysis of rat tissues under physiological and toxicological conditions

In biological research the analysis of gene expression levels in cells and tissues can be a powerful tool to gain insights into biological processes. For this, quantitative RT-PCR (RT-qPCR) is a popular method that often involve the use of constitutively expressed endogenous reference (or ‘housekeeping’) gene for normalization of data. Thus, it is essential to use reference genes that have been verified to be stably expressed within the specific experimental setting. Here, we have analysed the expression stability of 12 commonly used reference genes (Actb, B2m, Gapdh, Hprt, Pgk1, Rn18s, Rpl13a, Rps18, Rps29, Sdha, Tbp and Ubc) across several juvenile and adult rat tissues (liver, adrenal, prostate, fat pad, testis and ovaries), both under normal conditions and following exposure to various chemicals during development. Employing NormFinder and BestKeeper softwares, we found Hprt and Sdha to be amongst the most stable genes across normal and manipulated tissues, with several others also being suitable for most tissues. Tbp and B2m displayed highest variability in transcript levels between tissues and developmental stages. It was also observed that the reference genes were most unstable in liver and testis following toxicological exposure. For future studies, we propose the use of more than one verified reference gene and the continuous monitoring of their suitability under various experimental conditions, including toxicological studies, based on changes in threshold (Ct) values from cDNA samples having been reverse-transcribed from a constant input concentration of RNA.

Nanoparticulate silver increases uric acid and allantoin excretion in rats, as identified by metabolomics

Metabolomic investigation of rat urine was employed to identify mammalian metabolites affected by ionic or nanoparticulate silver. Female and male Wistar rats were administered silver nanoparticles (2.25, 4.5 or 9.0 mg kg−1 body weight per day) or ionic silver (silver acetate, 9.0 mg silver kg−1 bw per day) by oral gavage for 28 days. On day 18, urine was collected for 24 h and subjected to metabolomics with high performance liquid chromatography–quadropole time‐of‐flight mass spectrometry (HPLC‐QTOF‐MS)‐based separation and detection. Principal component analysis was subsequently applied to the data. Metabolomic differences in urine composition were found in female rats but not in male rats. Several metabolites were identified by the use of elemental composition calculated from the exact mass combined with searches in the Human Metabolome Database.The metabolite identities were eventually verified by co‐chromatography with authentic standards. Differences were found in uric acid and its degradation product, allantoin. Administration of nanoparticulate silver increased both metabolites, whereas ionic silver only increased allantoin. In conclusion, metabolomic investigation of rat urine showed that increased levels of uric acid and allantoin were associated with exposure to nanoparticulate silver. Copyright

Toxicological risk assessment of elemental gold following oral exposure to sheets and nanoparticles - A review.

Elemental gold is used as a food coloring agent and in dental fillings. In addition, gold nanoparticles are gaining increasing attention due to their potential use as inert carriers for medical purposes. Although elemental gold is considered to be inert, there is evidence to suggest the release of gold ions from its surface. Elemental gold, or the released ions, is, to some extent, absorbed in the gastrointestinal tract. Gold is distributed to organs such as the liver, heart, kidneys and lungs. The main excretion route of absorbed gold is through urine. Data on the oral toxicity of elemental gold is limited. The acute toxicity of elemental gold seems to be low, as rats were unaffected by a single dose of 2000mg nanoparticles/kg of body weight. Information on repeated dose toxicity is very limited. Skin rashes have been reported in humans following the ingestion of liquors containing gold. In addition, gold released from dental restorations has been reported to increase the risk of developing gold hypersensitivity. Regarding genotoxicity, in vitro studies indicate that gold nanoparticles induce DNA damage in mammalian cells. In vivo, gold nanoparticles induce genotoxic effects in Drosophila melanogaster; however, genotoxicity studies in mammals are lacking. Overall, based on the literature and taking low human exposure into account, elemental gold via the oral route is not considered to pose a health concern to humans in general.