Uptake, retention and internalization of quantum dots in Daphnia is influenced by particle surface functionalization.

Nanomaterials are a diverse group of compounds whose inevitable release into the environment warrants study of the fundamental processes that govern the ingestion, uptake and accumulation in aquatic organisms. Nanomaterials have the ability to transfer to higher trophic levels in aquatic ecosystems, and recent evidence suggests that the surface chemistry of both the nanoparticle and biological membrane can influence uptake kinetics. Therefore, our study investigates the effect of surface functionalization on uptake, internalization and depuration in Daphnia spp. Uncharged (polyethylene glycol; PEG), positively charged (amino-terminated: NH2) and negatively charged (carboxyl-modified; COOH) cadmium selenide/zinc sulfide quantum dots were used to monitor ingestion, uptake and depuration of nanometals in Daphnia magna and Ceriodaphnia dubia over 24h of exposure. These studies demonstrated that particles with higher negative charge (COOH quantum dots) were taken up to a greater extent by Daphnia (259.17±17.70 RFU/20 Daphnia) than either the NH2 (150.01±18.91) or PEG quantum dots (95.17±9.78), however this is likely related to the functional groups attached to the nanoparticles as there were no real differences in zeta potential. Whole body fluorescence associates well with fluorescent microscopic images obtained at the 24h timepoint. Confocal and electron microscopic analysis clearly demonstrated that all three types of quantum dots could cross the intestinal epithelial barrier and be translocated to other cells. Upon cessation of exposure, elimination of all three materials was biphasic with rapid initial clearance that likely represents elimination of material remaining in the GI tract followed by a much slower elimination phase that likely represents elimination of internalized material. These studies demonstrate that daphnids can take up intact nanomaterial from the water column and that this uptake is strongly influenced by particle surface functionalization. In addition, the usefulness of using quantum dots as a proxy for other nanometals (no acute toxicity, clear visualization in electron microscopy), in conjunction with several different imaging techniques in assessing uptake and accumulation of nanoparticles in daphnids was demonstrated.

Phenol-oxidizing laccases from the termite gut.

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

Neurological effects of acute uranium exposure with and without stress.

Circulating uranium rapidly enters the brain and may cause adverse effects on the nervous system that are potentially modulated by stress. In this study, the neurological effects of a single intramuscular injection of 0, 0.1, 0.3, or 1 mg uranium/kg (as uranyl acetate, UA) in rats were examined in the presence and absence of stress. Treatment with UA produced time and dose-dependent increases in serum and regional brain uranium levels. While serum levels returned to control levels by day 30, brain levels remained elevated. Application of stress did not affect the distribution or retention of uranium. Exposure to 1 mg U/kg significantly decreased ambulatory activity, weight gain, forelimb grip strength and transiently impaired working memory. Effects on grip strength and memory were prevented by application of stress prior to uranium exposure. Striatal dopamine content was reduced by 30% 3 days after treatment with 1mg/kg (59+/-6 nmol/mg tissue versus 41+/-5 nmol/mg tissue), but levels returned to control 7 days after uranium exposure. The effect on dopamine was ameliorated by prior application of stress. Exposure to UA did not alter 3,4 dihydroxyphenylacetic acid (DOPAC) levels or numbers of D2 receptors in the striatum. No effect of uranium or stress was observed on levels of GABA, serotonin, norepinephrine, or glutathione (GSH) in the striatum, hippocampus, cerebellum, or cortex. These results indicate that single intramuscular exposures to uranium produce sustained elevation of brain uranium levels and at doses above 0.3 mg/kg can have adverse neurological effects. Application of stress prior to uranium administration modulates neurological effects, but the mechanism is not due to effects on uranium distribution. Uranium exposure also produced renal toxicity which must be considered to accurately assess the effects of uranium on neurological function.

Esterase inhibition in SH-SY5Y human neuroblastoma cells following exposure to organophosphorus compounds for 28 days.

Esterase inhibition was determined in SH-SY5Y human neuroblastoma cells grown in serum-free media and exposed to 10(-11) to 10(-7) M concentrations of organophosphorus (OP) compounds for 28 days. To examine metabolic activation in these exposures, pairs of pro- and active toxicants were studied, including chlorpyrifos and its oxon, parathion and paraoxon, and tri-ortho-tolyl phosphate and phenyl saligenin phospahte. Inhibition of acetylcholinesterase was greater in cells treated for 28 days with all active organophosphorus compounds than it was in cells treated only once with the same concentration of a given OP compound. The protoxicants chlorpyrifos and parathion produced acetylcholinesterase inhibition after multiple exposures although no inhibition was seen following a single exposure to these agents. Exacerbation of neurotoxic esterase inhibition by multiple exposures to the test compounds was not as pronounced as that of acetylcholinesterase. Exposure to the test compounds for 28 days did not significantly enhance esterase inhibition produced by a subsequent exposure to 10(-9) M chlorpyrifos-oxon. The results indicate that in vitro methods can be used to study the effect of multiple OP exposures on esterase activity.

Determination of acrylamide and glycidamide in rat plasma by reversed-phase high performance liquid chromatography.

Acrylamide is a widely used monomer that produces peripheral neuropathy. It is metabolized to the epoxide, glycidamide, which is also considered to be neurotoxic. A new reversed-phase high-performance liquid chromatography (HPLC) method is described that permits simultaneous determination of acrylamide and glycidamide in rat plasma. Samples were deproteinized with acetonitrile and chromatography was performed using isocratic elution and UV absorption detection. The limits of detection for acrylamide and glycidamide were 0.05 and 0.25 microg/ml in plasma, respectively, and recovery of both analytes was greater than 90%. The assay was linear from 0.1 to 100 microg/ml for acrylamide and from 0.5 to 100 microg/ml for glycidamide. Variation over the range of the standard curve was less than 15%. The method was used to determine the concentration-time profiles of acrylamide and glycidamide in the plasma of acrylamide-treated rats.

Metabolism, toxicokinetics and hemoglobin adduct formation in rats following subacute and subchronic acrylamide dosing.

Long-term, low-dose (subchronic) oral acrylamide (ACR) exposure produces peripheral nerve axon degeneration, whereas irreversible axon injury is not a component of short-term, higher dose (subacute) i.p. intoxication [Toxicol Appl Pharmacol 1998;151:211]. It is possible that this differential axonopathic expression is a product of exposure-dependent differences in ACR biotransformation and/or tissue distribution. Therefore, we determined the toxicokinetics and metabolism of ACR following subchronic oral (2.8 mM in drinking water for 34 days) or subacute i.p. (50 mg/kg per day for 11 days) administration to rats. Both dosing regimens produced moderate levels of behavioral neurotoxicity and, for each, ACR was rapidly absorbed from the site of administration and evenly distributed to tissues. Peak ACR plasma concentrations and tissue levels were directly related to corresponding daily dosing rates (20 or 50 mg/kg per day). During subchronic oral dosing a larger proportion (30%) of plasma ACR was converted to the epoxide metabolite glycidamide (GLY) than was observed following subacute i.p. intoxication (8%). This subchronic effect was not specifically related to changes in enzyme activities involved in GLY formation (cytochrome P450 2E1) ormetabolism (epoxide hydrolases). Both ACR and GLY formed hemoglobin adducts during subacute and subchronic dosing, the absolute quantity of which did not change as a function of neurotoxicant exposure. Compared to subacute i.p. exposure, the subchronic schedule produced approximately 30% less ACR adducts but two-fold more GLY adducts. GLY has been considered to be an active ACR metabolite and might mediate axon degeneration during subchronic ACR administration. However, corresponding peak GLY plasma concentrations were relatively low and previous studies have shown that GLY is only a weak neurotoxicant. Our study did not reveal other toxicokinetic idiosyncrasies that might be a basis for subchronic induction of irreversible axon damage. Consequently the mechanism of axon degeneration does not appear to involve route- or rate-dependent differences in metabolism or disposition.

Nerve conduction and ATP concentrations in sciatic-tibial and medial plantar nerves of hens given phenyl saligenin phosphate.

To assess the relationship of nerve conduction and adenosine triphosphate (ATP) status in organophosphorus-induced delayed neuropathy (OPIDN), we evaluated both in adult hen peripheral nerves following exposure to a single 2.5 mg/kg dose of phenyl saligenin phosphate (PSP). ATP concentrations were determined at days 2, 4, 7, and 14 post-dosing, from five segments (n = 5 per group) representing the entire length of the sciatic-tibial and medial plantar nerve. Initial effects of PSP dosing were seen in the most distal segment at day 2, when a transient ATP concentration increase (388 +/- 79 pmol/ml/mg versus control value of 215 +/- 23, P < 0.05) was noted. Subsequently, ATP concentration in this distal segment returned to normal. In the most proximal nerve segment, ATP concentrations were decreased on day 7, and further decreased on day 14 post-dosing (P < 0.05). Changes in ATP concentration and nerve conduction velocity begin at post-dosing day 2, and were found prior to development of clinical neuropathy and axonopathic lesions. These results suggest that alterations in sciatic-tibial and medial plantar nerve conduction associated with sciatic-tibial and medial plantar nerve ATP concentration are early events in the development of OPIDN.

In vitro tissue specificity for arsine and arsenite toxicity in the rat.

The mechanism of arsine (AsH3) toxicity is not completely understood. In this investigation, we determined AsH3 and arsenite (AsIII) toxicity in Sprague Dawley rat blood, liver, and kidney. In all systems, there were dose- and time-dependent responses. Red blood cells were very susceptible to AsH3 toxicity. This was demonstrated by an immediate intracellular potassium loss and by hemolysis and lactate dehydrogenase (LDH) leakage that occurred by one h. AsIII concentrations up to 1 mM were not toxic to red blood cells using these indicators. Both AsH3 and AsIII produced toxicity in primary hepatocytes. Both produced significant LDH leakage and decreases in intracellular K+ by 5 h, but AsIII was more toxic than AsH3. At 24 h, both arsenic species showed similar toxicity. In renal cortical epithelial cells, AsH3 produced no effects on LDH and K+ over a 5-h period but produced significant LDH leakage by 24 h. In these cells, AsIII produced significant toxicity as early as in 3 h. These results showed that unchanged AsH3 produced toxicity in tissues, in addition to blood, and that toxicity of arsenicals is arsenic species- and tissue-dependent.

Modulation of bronchial epithelial cell barrier function by in vitro jet propulsion fuel 8 exposure.

The loss of epithelial barrier integrity in bronchial and bronchiolar airways may be an initiating factor in the observed onset of toxicant-induced lung injuries. Acute 1-h inhalation exposures to aerosolized jet propulsion fuel 8 (JP-8) have been shown to induce cellular and morphological indications of pulmonary toxicity that was associated with increased respiratory permeability to 99mTc-DTPA. To address the hypothesis that JP-8 jet fuel-induced lung injury is initiated through a disruption in the airway epithelial barrier function, paracellular mannitol flux of BEAS-2B human bronchial epithelial cells was measured. Incubation of confluent cell cultures with non-cytotoxic concentrations of JP-8 or n-tetradecane (C14), a primary constituent of JP-8, for a 1-h exposure period resulted in dose-dependent increases of paracellular flux. Following exposures of 0.17, 0.33, 0.50, or 0.67 mg/ml, mannitol flux increased above vehicle controls by 10, 14, 29, and 52%, respectively, during a 2-h incubation period immediately after each JP-8 exposure. C14 caused greater mannitol flux increases of 37, 42, 63, and 78%, respectively, following identical exposure conditions. The effect on transepithelial mannitol flux reached a maximum at 12 h and spontaneously reversed to control values over a 48-h recovery period, for both JP-8 and C14 exposure. These data indicate that non-cytotoxic exposures to JP-8 or C14 exert a noxious effect on bronchial epithelial barrier function that may preclude pathological lung injury.

Glutathione, albumin, cysteine, and cys-gly effects on toxicity and accumulation of mercuric chloride in LLC-PK1 cells.

Speciation plays a profound if not dominant role in both transport and toxicity of Hg(II). Hg(II) has a high affinity for sulfhydryl groups. The formation constant for Hg2+ and the anionic form of a sulfhydryl group R-S- is > or =10(10) higher than that for the carboxyl or amino groups. The kidneys are the target organ for Hg(II) toxicity and the primary site of Hg(II) accumulation. Sulfhydryl groups have been implicated in both transport and nephrotoxicity; however, the role endogenous thiol compounds play in these parameters is not clear. The roles that albumin, glutathione, and the glutathione-derived complexes cysteinylglycine and L-cysteine play in toxicity and accumulation of HgCl2 were studied in LLC-PK1 cells incubated with different Hg(II):thiol ratios. In cysteine-containing medium, almost all 1:2 Hg(II):thiol complexes protected against Hg(II) toxicity up to 120 microM Hg, increased membrane-bound Hg(II), and decreased intracellular Hg(II) accumulation. In cysteine-free medium, all 1:1 Hg(II):thiol complexes were as toxic as uncomplexed Hg(II), and almost all 1:2 Hg(II):thiol complexes protected at > or =20 microM Hg, except albumin, which protected at < or =20 microM Hg. In cysteine-free but cystine-containing medium, two 1:1 Hg(II):thiol complexes were toxic at > or =80 microM Hg and two provided complete protection. All 1:2 complexes provided protection at 80-160 microM Hg. This investigation used defined media to demonstrate that mercury cytotoxicity in LLC-PK1 cells was dependent on Hg(II) concentration, the ligand, and the presence of a cysteine source for the cells. These effects were only partially explained by intracellular Hg(II) levels.

Effects of micronutrients on metal toxicity.

There is growing evidence that micronutrient intake has a significant effect on the toxicity and carcinogenesis caused by various chemicals. This paper examines the effect of micronutrient status on the toxicity of four nonessential metals: cadmium, lead, mercury, and arsenic. Unfortunately, few studies have directly examined the effect of dietary deficiency or supplementation on metal toxicity. More commonly, the effect of dietary alteration must be deduced from the results of mechanistic studies. We have chosen to separate the effect of micronutrients on toxic metals into three classes: interaction between essential micronutrients and toxic metals during uptake, binding, and excretion; influence of micronutrients on the metabolism of toxic metals; and effect of micronutrients on secondary toxic effects of metals. Based on data from mechanistic studies, the ability of micronutrients to modulate the toxicity of metals is indisputable. Micronutrients interact with toxic metals at several points in the body: absorption and excretion of toxic metals; transport of metals in the body; binding to target proteins; metabolism and sequestration of toxic metals; and finally, in secondary mechanisms of toxicity such as oxidative stress. Therefore, people eating a diet deficient in micronutrients will be predisposed to toxicity from nonessential metals.

Sequence of toxic events in arsine-induced hemolysis in vitro: implications for the mechanism of toxicity in human erythrocytes.

Arsine, the hydride of arsenic (AsH3), is the most acutely toxic form of arsenic, causing rapid and severe hemolysis upon exposure. The mechanism of action is not known, and there are few detailed investigations of the toxicity in a controlled system. To examine arsine hemolysis and understand the importance of various toxic responses, human erythrocytes were incubated with arsine in vitro, and markers of toxicity were determined as a function of time. The earliest indicators of damage were changes in sodium and potassium levels. Within 5 min incubation with 1 mm arsine, the cells lost volume control, manifested by leakage of potassium, influx of sodium, and increases in hematocrit. Arsine did not, however, significantly alter ATP levels nor inhibit ATPases. These changes were followed by profound disturbances in membrane ultrastructure (examined by light and electron microscopy). By 10 min, significant numbers of damaged cells formed, and their numbers increased over time. These events preceded hemolysis, which was not significant until 30 min. It has been proposed that arsine interacts with hemoglobin to form toxic hemoglobin oxidation products, and this was also investigated as a potential cause of hemolysis. Essentially on contact with arsine, methemoglobin was formed but only reached 2-3% of the total cellular hemoglobin and remained unchanged for up to 90 min. There was no evidence that further oxidation products (hemin and Heinz bodies) were formed in this system. Based on these observations, hemolysis appears to be dependent on membrane disruption by a mechanism other than hemoglobin oxidation.