Search

Toxaphene is an organochlorine pesticide and environmental contaminant that is concerning due to its atmospheric transport and persistence in soil. In Florida, toxaphene and other organochlorine pesticides were used heavily in agriculture on the north shore of Lake Apopka and they are still detectable in soil. Wild largemouth bass that inhabit the lake and the marshes along the north shore have been exposed to a variety of organochlorine pesticides including dieldrin, methoxychlor, and p,p′-DDE, among others. While these other organochlorine pesticides have been studied for their endocrine disrupting effects in largemouth bass, there is little information for toxaphene. In this study, male and female largemouth bass were given food containing 50 mg/kg toxaphene for almost 3 months, to achieve tissue levels similar to those found in fish at Lake Apopka. Sex-specific toxicity was then evaluated by measuring various reproductive endpoints and transcriptomic changes. In females, gonadosomatic index showed a trend towards reduction (p = 0.051) and plasma vitellogenin was reduced by ~40% relative to controls. However plasma levels of 17β-estradiol and testosterone were not perturbed by toxaphene exposure. These data suggest that toxaphene does not act as a weak estrogen as many other organochlorine pesticides do, but rather appears to be acting as an antiestrogen in female fish. There were no obvious changes in the gonadosomatic index and plasma hormones in male bass. However, ex vivo explant experiments revealed that toxaphene prevented human chorionic gonadotropin-stimulated testosterone production in the testis. This suggested that toxaphene had anti-androgenic effects in males. Subsequent transcriptomic analyses of the testis revealed that androgen receptor/beta-2-microglobulin signaling was up-regulated while insulin-related pathways were suppressed with toxaphene, which could be interpreted as a compensatory response to androgen suppression. In the male liver, the transcriptome analysis revealed an overwhelming suppression in immune-related signaling cascades (e.g. lectin-like receptor and ITSM-Containing Receptor signaling, CD16/CD14 Proinflammatory Monocyte Activation, and CD38/CD3-JUN/FOS/NF-kB Signaling in T-cell Proliferation). Overall, this study showed that toxaphene induced sex-specific effects. The transcriptomic and physiological responses observed can contribute to the development of adverse outcome pathways for toxaphene exposure in fish.

Cadmium is a heavy metal that can accumulate to toxic levels in the environment leading to detrimental effects in animals and humans including kidney, liver and lung injuries. Using a transcriptomics approach, genes and cellular pathways affected by a low dose of cadmium were investigated. Adult largemouth bass were intraperitoneally injected with 20μg/kg of cadmium chloride (mean exposure level - 2.6μg of cadmium per fish) and microarray analyses were conducted in the liver and testis 48h after injection. Transcriptomic profiles identified in response to cadmium exposure were tissue-specific with the most differential expression changes found in the liver tissues, which also contained much higher levels of cadmium than the testis. Acute exposure to a low dose of cadmium induced oxidative stress response and oxidative damage pathways in the liver. The mRNA levels of antioxidants such as catalase increased and numerous transcripts related to DNA damage and DNA repair were significantly altered. Hepatic mRNA levels of metallothionein, a molecular marker of metal exposure, did not increase significantly after 48h exposure. Carbohydrate metabolic pathways were also disrupted with hepatic transcripts such as UDP-glucose, pyrophosphorylase 2, and sorbitol dehydrogenase highly induced. Both tissues exhibited a disruption of steroid signaling pathways. In the testis, estrogen receptor beta and transcripts linked to cholesterol metabolism were suppressed. On the contrary, genes involved in cholesterol metabolism were highly increased in the liver including genes encoding for the rate limiting steroidogenic acute regulatory protein and the catalytic enzyme 7-dehydrocholesterol reductase. Integration of the transcriptomic data using functional enrichment analyses revealed a number of enriched gene networks associated with previously reported adverse outcomes of cadmium exposure such as liver toxicity and impaired reproduction.

Exposure to excessive levels of manganese (Mn) is associated with the development of movement disorders, with symptoms overlapping with Parkinson's disease. Oxidative damage has been implicated as a key contributor to Mn-induced neurotoxicity. We have recently reported that divalent Mn (Mn(2+)) stimulates brain microglia to produce and release hydrogen peroxide (H2O2), and microglial-free radical generation facilitates Mn(2+)-induced dopaminergic neurotoxicity. The goal of this study was to elucidate the underlying mechanism of the Mn(2+)-induced H2O2 production in microglia. Exposure to low micromolar concentrations of Mn(2+), but not divalent copper, cadmium, nickel, cobalt, zinc, and iron, induced a significant production of H2O2 from rat microglial but not astroglial cells. Subcellular fractionation studies revealed that Mn(2+) was capable of inducing significant H2O2 production in the mitochondrial but not the cytosolic or nuclear fraction prepared from microglia. Analysis of the relative contribution of mitochondrial respiratory chain complexes indicated that Mn(2+)-induced mitochondrial H2O2 production required the presence of complex II substrate succinate. In contrast, complex I substrates malate and glutamate failed to support H2O2 production in the presence of Mn(2+). Furthermore, the succinate-supported Mn(2+)-induced mitochondrial H2O2 production was abolished by pharmacological inhibition of complex II but not that of complexes I and III, suggesting that mitochondrial complex II is a key mediator in Mn(2+)-induced H2O2 production. These findings advance our knowledge on the mechanisms by which Mn induces oxidative stress and the potential contribution to Mn neurotoxicity.

Largemouth bass (Micropterus salmoides) inhabiting Lake Apopka, Florida are exposed to high levels of persistent organochlorine pesticides (OCPs) and dietary uptake is a significant route of exposure for these apex predators. The objectives of this study were to determine the dietary effects of two organochlorine pesticides (p, p'-dichlorodiphenyldichloroethylene; p, p' DDE and methoxychlor; MXC) on the reproductive axis of largemouth bass. Reproductive bass (late vitellogenesis) were fed one of the following diets: control pellets, 125ppm p, p'-DDE, or 10ppm MXC (mg/kg) for 84days. Due to the fact that both p,p' DDE and MXC have anti-androgenic properties, the anti-androgenic pharmaceutical flutamide was fed to a fourth group of largemouth bass (750ppm). Following a 3 month exposure, fish incorporated p,p' DDE and MXC into both muscle and ovary tissue, with the ovary incorporating 3 times more organochlorine pesticides compared to muscle. Endpoints assessed were those related to reproduction due to previous studies demonstrating that these pesticides impact the reproductive axis and we hypothesized that a dietary exposure would result in impaired reproduction. However, oocyte distribution, gonadosomatic index, plasma vitellogenin, and plasma sex steroids (17β-estradiol, E2 and testosterone, T) were not different between control animals and contaminant-fed largemouth bass. Moreover, neither p, p' DDE nor MXC affected E2 or T production in ex vivo oocyte cultures from chemical-fed largemouth bass. However, both pesticides did interfere with the normal upregulation of androgen receptor that is observed in response to human chorionic gonadotropin in ex vivo cultures, an observation that may be related to their anti-androgenic properties. Transcriptomics profiling in the ovary revealed that gene networks related to cell processes such as leukocyte cell adhesion, ossification, platelet function and inhibition, xenobiotic metabolism, fibrinolysis, and thermoregulation were altered by p, p' DDE, MXC, and flutamide. Interestingly, immune-related gene networks were suppressed by all three chemicals. The data suggest that p, p' DDE and flutamide affected more genes in common with each other than either chemical with MXC, consistent with studies suggesting that p, p' DDE is a more potent anti-androgen than MXC. These data demonstrate that reproductive health was not affected by these specific dietary treatments, but rather the immune system, which may be a significant target of organochlorine pesticides. The interaction between the reproductive and immune systems should be considered in future studies on these legacy and persistent pesticides.

Adsorption of natural organic matter (NOM) on nanoparticles can have dramatic impacts on particle dispersion resulting in altered fate and transport as well as bioavailability and toxicity. In this study, the adsorption of Suwannee River humic acid (SRHA) on silver nanoparticles (nano-Ag) was determined and showed a Langmuir adsorption at pH 7 with an adsorption maximum of 28.6 mg g(-1) nano-Ag. It was also revealed that addition of

α,β-Unsaturated carbonyls make up an important class of chemicals involved in environmental toxicity and disease processes. Whereas adduction of cysteine residues on proteins is a well-documented reaction of these chemicals, such a generic effect cannot explain the molecular mechanism of cytotoxicity. Instead, more detailed information is needed regarding the possible specificity and kinetics of cysteine targeting and the quantitative relationship between adduct burden and protein dysfunction. To address these data gaps, we incubated purified human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with acrylamide (ACR), acrolein, or methylvinyl ketone (MVK). Results show that these α,β-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration- and time-dependent manner. The rank order of enzyme inhibition (K(I)) (i.e., ACR ≪ MVKacrolein) was related to the calculated electrophilic reactivity of each compound and to the corresponding kinetics of cysteine adduct formation. Tandem mass spectrometry revealed that adduct formation was selective at lower concentrations; i.e., ACR preferentially formed adducts with Cys152 (residues 146-162). At higher concentrations, ACR also formed adducts with Cys156 and Cys247 (residues 235-248). Adduct formation at Cys152 was correlated to enzyme inhibition, which is consistent with the regulatory role of this residue in enzyme function and its location within the GAPDH active site. Further analyses indicated that Cys152 was present in a pK(a)-lowering microenvironment (pK(a) = 6.03), and at physiological pH, the corresponding sulfhydryl group exists in the highly reactive nucleophilic thiolate state. These data suggest a general cytotoxic mechanism in which electrophilic α,β-unsaturated carbonyls selectively form adducts with reactive nucleophilic cysteine residues specifically associated with the active sites of proteins. These specialized cysteine residues are toxicologically relevant molecular targets, because chemical derivatization causes loss of protein function.

Many chemical toxicants and/or their active metabolites are electrophiles that cause cell injury by forming covalent bonds with nucleophilic targets on biological macromolecules. Covalent reactions between nucleophilic and electrophilic reagents are, however, discriminatory since there is a significant degree of selectivity associated with these interactions. Over the course of the past few decades, the theory of Hard and Soft, Acids and Bases (HSAB) has proven to be a useful tool in predicting the outcome of such reactions. This concept utilizes the inherent electronic characteristic of polarizability to define, for example, reacting electrophiles and nucleophiles as either hard or soft. These HSAB definitions have been successfully applied to chemical-induced toxicity in biological systems. Thus, according to this principle, a toxic electrophile reacts preferentially with biological targets of similar hardness or softness. The soft/hard classification of a xenobiotic electrophile has obvious utility in discerning plausible biological targets and molecular mechanisms of toxicity. The purpose of this perspective is to discuss the HSAB theory of electrophiles and nucleophiles within a toxicological framework. In principle, covalent bond formation can be described by using the properties of their outermost or frontier orbitals. Because these orbital energies for most chemicals can be calculated using quantum mechanical models, it is possible to quantify the relative softness (σ) or hardness (η) of electrophiles or nucleophiles and to subsequently convert this information into useful indices of reactivity. This atomic level information can provide insight into the design of corroborative laboratory research and thereby help investigators discern corresponding molecular sites and mechanisms of toxicant action. The use of HSAB parameters has also been instrumental in the development and identification of potential nucleophilic cytoprotectants that can scavenge toxic electrophiles. Clearly, the difficult task of delineating molecular sites and mechanisms of toxicant action can be facilitated by the application of this quantitative approach.

Nanoparticles are being rapidly assimilated into numerous research fields and consumer products. A concurrent increase in human exposure to such materials is expected. Magnetic nanoparticles (MNPs) possess unique and beneficial features, increasing their functionality and integrative potential. However, MNP toxicity characterization is limited, especially in regards to the human respiratory system. This study aimed to assess the in vitro effects of airborne MNPs on BEAS-2B cells. Uncoated iron oxide was compared with two amorphous silica-coated MNPs, hypothesizing the coatings reduced toxicity and increased particle stability.BEAS-2B cells were cultured at an air-liquid interface and exposed to airborne MNPs using a fabricated exposure device. Indices of cytotoxicity, inflammatory response, oxidative stress, and iron homeostasis were monitored postexposure via cell viability assays and qRT-PCR. Concentrations of soluble iron-associated with different MNPs were also examined before and after contact with several aqueous organic and inorganic acids.The silica-coated MNPs had reduced soluble iron concentrations. This result indicates that the silica coating provides a barrier to and prevents the mobilization of soluble iron from the particle to the cell, thereby reducing the risk of oxidative stress or alterations of iron homeostasis. Cells exposed to MagSilica50 and MagSilica50-85® showed little to no indications of cytotoxicity or induction of inflammatory response/oxidative stress at the examined delivery concentrations.MNPs coated with amorphous silica are protected from acidic erosion. Correspondingly, the particle stability translates into reduced cytotoxicity and cellular influence on human airway epithelial cells.

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.

Exposure to dieldrin induces neurotoxic effects in the vertebrate CNS and disrupts reproductive processes in teleost fish. Reproductive impairment observed in fish by dieldrin is likely the result of multiple effects along the hypothalamic-pituitary-gonadal axis but the molecular signaling cascades are not well characterized. To better elucidate the mode of action of dieldrin in the hypothalamus, this study measured neurotransmitter levels and examined the transcriptomic response in female largemouth bass (LMB) to an acute treatment of dieldrin. Male and female LMB were injected with either vehicle or 10 mg dieldrin/kg and sacrificed after seven days. There were no significant changes in dopamine or DOPAC concentrations in the neuroendocrine brain of males and females after treatment but GABA levels in females were moderately increased 20–30% in the hypothalamus and cerebellum. In the female hypothalamus, there were 227 transcripts (p

Synaptic dysfunction appears to be an early pathogenic event in Alzheimer's disease, amyotrophic lateral sclerosis and Parkinson's disease. Although the molecular mechanism of this synaptotoxicity is not known, evidence suggests that these diseases are characterized by a common pathophysiological cascade involving oxidative stress, lipid peroxidation and the subsequent liberation of alpha,beta-unsaturated carbonyl derivatives such as acrolein and 4-hydroxy-2-nonenal (HNE). A diverse body of in vivo and in vitro data have shown that these soft electrophilic chemicals can cause nerve terminal damage by forming Michael-type adducts with nucleophilic sulfhydryl groups on presynaptic proteins. Therefore, the endogenous generation of acrolein and HNE in oxidatively stressed neurons of certain brain regions might be mechanistically related to the synaptotoxicity associated with neurodegenerative conditions. In addition, acrolein and HNE are members of a large class of structurally related chemicals known as the type-2 alkenes. Chemicals in this class (e.g., acrylamide, methylvinyl ketone, and methyl acrylate) are pervasive pollutants in human environments and new research has shown that these alpha,beta-unsaturated carbonyl derivatives are also toxic to nerve terminals. In this review, we provide evidence that the regional synaptotoxicity, which develops during the early stages of many neurodegenerative diseases, is mediated by endogenous generation of acrolein and HNE. Based on a presumed common nerve terminal site of action, we propose that the onset and progression of this neuropathogenic process is accelerated by environmental exposure to other type-2 alkenes.

Conjugated alpha,beta-unsaturated carbonyl derivatives such acrylamide, acrolein, and 4-hydroxy-2-nonenal (HNE) are members of a large class of chemicals known as the type-2 alkenes. Human exposure through diet, occupation, and pollution is pervasive and has been linked to toxicity in most major organs. Evidence suggests that these soft electrophiles produce toxicity by a common mechanism involving the formation of Michael-type adducts with nucleophilic sulfhydryl groups. In this commentary, the adduct chemistry of the alpha,beta-unsaturated carbonyls and possible protein targets will be reviewed. We also consider how differences in electrophilic reactivity among the type-2 alkenes impact corresponding toxicokinetics and toxicological expression. Whereas these concepts have mechanistic implications for the general toxicity of type-2 alkenes, this commentary will focus on the ability of these chemicals to produce presynaptic damage via protein adduct formation. Given the ubiquitous environmental presence of the conjugated alkenes, discussions of molecular mechanisms and possible neurotoxicological risks could be important. Understanding the neurotoxicodynamic of the type-2 alkenes might also provide mechanistic insight into neurodegenerative conditions where neuronal oxidative stress and presynaptic dysfunction are presumed initiating events. This is particularly germane to a recent proposal that lipid peroxidation and the subsequent liberation of acrolein and HNE in oxidatively stressed neurons mediate synaptotoxicity in brains of Alzheimer's disease patients. This endogenous neuropathogenic process could be accelerated by environmental type-2 alkene exposure because common nerve terminal proteins are targeted by alpha,beta-unsaturated carbonyl derivatives. Thus, the protein adduct chemistry of the conjugated type-2 alkenes offers a mechanistic explanation for the environmental toxicity induced by these chemicals and might provide insight into the pathogenesis of certain human neurodegenerative diseases.

Military use of depleted uranium (DU) has renewed interest in the toxicology of this metal. In this study, the nephrotoxicity of single exposure DU was assessed with and without pre-exposure stress. Adult male Sprague–Dawley rats (n = 288) were administered a single IM dose of 0, 0.1, 0.3 or 1.0 mg/kg DU. Corticosterone concentrations (ng/ml, mean ± SD) were 763.65 ± 130.94 and 189.80 ± 90.81 for swim stressed and unstressed rats. Serum and kidney uranium concentration, hematocrit, chemistry, and renal histology were assessed on sacrifice days 1, 3, 7 and 30 post-DU-dosing. Dose related increases in serum and kidney uranium were noted. DU concentration peaked day 1 in the kidney and days 3–7, in the serum. Dose-related elevations of Cr and BUN concentrations were seen on days 3 and 7. A decline in serum albumin coincided with Cr and BUN suggesting protein losing nephropathy. Dose related acute tubular necrosis and proliferative glomulonephritis were seen. Tubular regeneration in low dose rats was almost complete by day 30. High dose rats had extensive tubular necrosis and delayed regeneration with focal residual chronic interstitial nephritis and cortical scarring. Glomular changes were reversed in all treatment groups by day 30. Stress exposure had no impact on any measured renal parameter.

Increasing use of metallic nanomaterials is likely to result in release of these particles into aqueous environments; however, it is unclear if these materials present a hazard to aquatic organisms. Because some dissolution of metal particles will occur, it is important to distinguish effects of nanoparticulates from dissolved metals. To address this issue, acute toxicity of soluble copper and 80 nm copper nanoparticle suspensions were examined in zebrafish. The results demonstrate that nanocopper is acutely toxic to zebrafish, with a 48 h LC50 concentration of 1.5 mg/L. Rapid aggregation of copper nanoparticles occurred after suspension in water, resulting in 50-60% of added mass leaving the water column. While dissolution of particulate copper occurred, it was insufficient to explain the mortality in nanocopper exposures. Histological and biochemical analysis revealed that the gill was the primary target organ for nanocopper. To further investigate the effects of nanocopper on the gill, zebrafish were exposed to 100 microg/L of nanocopper or to the concentration of soluble copper matching that present due to dissolution of the particles. Under these conditions, nanocopper produced different morphological effects and global gene expression patterns in the gill than soluble copper, clearly demonstrating that the effects of nanocopper on gill are not mediated solely by dissolution.

We have hypothesized that acrylamide (ACR) intoxication causes cumulative nerve terminal damage by forming adducts with nucleophilic cysteine sulfhydryl groups on critical presynaptic proteins. To determine the cumulative effects of ACR on the cysteine-containing proteome of nerve terminal, we employed cleavable isotope-coded affinity tagging (ICAT) and liquid chromatography-tandem mass spectrometry. ICAT analysis uses a sulfhydryl-specific tag to identify and quantitate cysteine-containing proteins. Synaptosomes were prepared from striatum of ACR-intoxicated rats (21 mg/kg/day × 7, 14, or 21 days) and their age-matched controls. The synaptosomal proteins of each experimental group were labeled with either light ( 12 C 9 —control) or heavy ( 13 C 9 —ACR) ICAT reagent. Results show that ACR intoxication caused a progressive reduction in the ICAT labeling of many nerve terminal proteins. A label-free mass spectrometric approach (multidimensional protein identification) was used to show that the observed reductions in ICAT incorporation were not due to general changes in protein abundance and that ACR formed adducts with cysteine residues on peptides which also exhibited reduced ICAT incorporation. The decrease in labeling was temporally correlated to the development of neurological toxicity and confirmed previous findings that cysteine adducts of ACR accumulate as a function of exposure. The accumulation of adduct is consistent with the cumulative neurotoxicity induced by ACR and suggests that cysteine adduct formation is a necessary neuropathogenic step. Furthermore, our analyses identified specific proteins (e.g., v-ATPase, dopamine transporter, N-ethylmaleimide-sensitive factor) that were progressively and significantly adducted by ACR and might, therefore, be neurotoxicologically relevant targets.

Lake Apopka (FL, USA) has elevated levels of some organochlorine pesticides in its sediments and a portion of its watershed has been designated a US Environmental Protection Agency Superfund site. This study assessed reproductive endpoints in Florida largemouth bass (LMB) ( Micropterus salmoides floridanus ) after placement into experimental ponds adjacent to Lake Apopka. LMB collected from a clean reference site (DeLeon Springs) were stocked at two periods of time into ponds constructed in former farm fields on the north shore of the lake. LMB were stocked during early and late oogenesis to determine if there were different effects of contamination on LMB that may be attributed to their reproductive stage. LMB inhabiting the ponds for ~ 4 months had anywhere from 2 to 800 times higher contaminant load for a number of organochlorine pesticides (e.g. p , p ′-DDE, methoxychlor) compared to control animals. Gonadosomatic index and plasma vitellogenin were not different between reproductively-stage matched LMB collected at reference sites compared to those inhabiting the ponds. However, plasma 17β-estradiol was lower in LMB inhabiting the Apopka ponds compared to ovary stage-matched LMB from the St. Johns River, a site used as a reference site. Sub-network enrichment analysis revealed that genes related to reproduction (granulosa function, oocyte development), endocrine function (steroid metabolism, hormone biosynthesis), and immune function (T cell suppression, leukocyte accumulation) were differentially expressed in the ovaries of LMB placed into the ponds. These data suggest that (1) LMB inhabiting the Apopka ponds showed disrupted reproduction and immune responses and that (2) gene expression profiles provided site-specific information by discriminating LMB from different macro-habitats.

The mitochondrial environment is rich in reactive oxygen species (ROS) that may ultimately peroxidize membrane proteins and generate unsaturated aldehydes such as 4-hydroxy-2-nonenal (4HNE). We had previously demonstrated the presence of hGSTA4-4, an efficient catalyst of 4HNE detoxification, in human liver mitochondria to the exclusion of the cytosol. In the present study, GSH-affinity chromatography was used in conjunction with biochemical and proteomic analysis to determine the presence of additional cytosolic glutathione S -transferases (GSTs) in human hepatic mitochondria. HPLC-subunit analysis of GSH affinity-purified liver mitochondrial proteins indicated the presence of several potential mitochondrial GST isoforms. Electrospray ionization-mass spectrometry analysis of eluted mitochondrial GST subunits yielded molecular masses similar to those of hGSTP1, hGSTA1 and hGSTA2. Octagonal matrix-assisted laser desorption/ionization time of flight mass spectrometry and proteomics analysis using MS-FIT confirmed the presence of these three GST subunits in mitochondria, and HPLC analysis indicated that the relative contents of the mitochondrial GST subunits were hGSTA1 > hGSTA2 > hGSTP1. The mitochondrial localization of the alpha and pi class GST subunits was consistent with immunoblotting analysis of purified mitochondrial GST. Enzymatic studies using GSH-purified mitochondrial GST fractions demonstrated the presence of significant GST activity using the nonspecific GST substrate 1-chloro-2,4-dinitrobenzene (CDNB), as well as 4HNE, δ 5 -androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic α,β-unsaturated aldehyde produced during the breakdown of membrane lipids, exceeded that observed in liver cytosol. These observations are suggestive of a role of GST in protecting against mitochondrial injury during the secondary phase of oxidative stress, or modulation of 4HNE-mediated mitochondrial signaling pathways. However, other properties of mitochondrial GST, such as conjugation of environmental chemicals and binding of lipophilic non-substrate xenobiotics and endogenous compounds, remain to be investigated.

In this study, largemouth bass (LMB) were subchronically exposed to p,p'-DDE or dieldrin in their diet to evaluate the effect of exposure on expression of genes involved in reproduction and steroid homeostasis. Using real-time PCR, we detected a different gene expression pattern for each OCP, suggesting that they each affect LMB in a different way. We also detected a different expression pattern among sexes, suggesting that sexes are affected differently by OCPs perhaps reflecting the different adaptive responses of each sex to dysregulation caused by OCP exposure.

Long-term exposure to depleted uranium (DU) has been shown to increase brain uranium and alter hippocampal function; however, little is known about the short-term kinetics of DU in the brain. To address this issue, temporal and regional distribution of brain uranium was investigated in male Sprague-Dawley rats treated with a single intraperitoneal injection of 1 mg uranium/kg as uranyl acetate. Due to the inherent stress of combat and the potential for stress to alter blood-brain barrier permeability, the impact of forced swim stress on brain uranium distribution was also examined in this model. Uranium in serum, hippocampus, striatum, cerebellum, and frontal cortex was quantified by inductively coupled plasma-mass spectrometry (ICP-MS) at 8 h, 24 h, 7 d, and 30 d after exposure. Uranium entered the brain rapidly and was initially concentrated in hippocampus and striatum. While multiple phases of uranium clearance were observed, overall clearance was relatively slow and the uranium content of hippocampus, cerebellum, and cortex remained elevated for more than 7 d after a single exposure. Prior exposure to stress significantly reduced hippocampal and cerebellar uranium 24 h post-exposure and tended to reduce uranium in all brain regions 7 d after exposure. The application of stress appeared to increase brain uranium clearance, as initial tissue levels were similar in stressed and unstressed rats.

Evidence suggests that acrylamide (ACR) neurotoxicity is mediated by decreased presynaptic neurotransmitter release. Defective release might involve disruption of neurotransmitter storage, and therefore, we determined the effects of in vivo and in vitro ACR exposure on 3H-dopamine (DA) transport into rat striatal synaptic vesicles. Results showed that vesicular DA uptake was decreased significantly in rats intoxicated at either 50 mg/kg/day x 5 days or 21 mg/kg/day x 21 days. ACR intoxication also was accompanied by a reduction in KCl-evoked synaptosomal DA release, although consistent changes in presynaptic membrane transport were not observed. Silver stain and immunoblot analyses suggested that reduced vesicular uptake was not due to active nerve terminal degeneration or to a reduction in the synaptic vesicle content of isolated striatal synaptosomes. Nor did the in vivo presynaptic effects of ACR involve changes in synaptosomal glutathione concentrations. In vitro exposure of striatal vesicles showed that both ACR and two sulfhydryl reagents, N-ethylmaleimide (NEM) and iodoacetic acid (IAA), produced concentration-dependent decreases in 3H-DA uptake. Although ACR was significantly less potent than either NEM or IAA, all three chemicals caused comparable maximal inhibitions of vesicular uptake. Kinetic analysis of DA uptake showed that in vitro exposure to either ACR or NEM decreased V(max) and increased K(m). Determination of radiolabel efflux from 3H-DA-loaded vesicles indicated that in vitro ACR did not affect neurotransmitter retention. These data suggest that ACR impaired neurotransmitter uptake into striatal synaptic vesicles, possibly by interacting with sulfhydryl groups on functionally relevant proteins. The resulting disruption of neurotransmitter storage might mediate defective presynaptic release.

Previous studies have demonstrated that prior exposures to uranium can produce acquired resistance to uranium nephrotoxicity. In this study, the potential role for heat shock proteins (Hsps) in acquired resistance to uranium nephrotoxicity was explored. Pretreatment of male Sprague-Dawley rats with a conditioning dose of uranyl acetate (5 mg/kg, i.p.) was found to diminish the severity of proximal convoluted tubule necrosis and azotemia produced by a subsequent, higher uranyl acetate dose (10 mg/kg, i.p., 10 days after the conditioning dose). Kidney homogenates from rats euthanized at the end of the conditioning period were found to contain elevated levels of Hsp25, Hsp32, and Hsp70i, but not Hsc70. Immunochemical staining of renal sections for Hsp25 and Hsp70i revealed that these proteins were prominently expressed in tubular epithelial cells in uranyl acetate pretreated animals. Morphological characteristics and staining for proliferating cell nuclear antigen (PCNA) indicated that the cells expressing high levels of Hsps were regenerating. In RK3E and LLC-PK1 renal epithelial cells in culture, Hsp induction by thermal pretreatment did not afford protection from uranyl acetate cytotoxicity. Further, treatment of RK3E and LLC-PK1 cells with uranyl acetate did not result in induction of Hsps, as occurs with other nephrotoxic heavy metals. These observations suggest that while stress proteins are elevated in acquired resistance to uranyl acetate in vivo, they are not responsible for diminished uranium nephrotoxicity but are an epiphenomenon of tubular epithelial regeneration.

Cystamine, a small disulfide-containing chemical, is neuroprotective in a transgenic mouse and a Drosophila model of Huntington's disease (HD) and decreases huntingtin aggregates in an in vitro model of HD. The mechanism of action of cystamine in these models is widely thought to involve inhibition of transglutaminase mediated cross-linking of mutant huntingtin in the process of aggregate formation/stabilization. In this study we show that cystamine, both in vitro and in a transgenic mouse model of HD (R6/2), increases levels of the cellular antioxidant L-cysteine. Several oxidative stress markers increase in HD brain. We provide further evidence of oxidative stress in mouse HD by demonstrating compensatory responses in R6/2 HD brains. We found age-dependent increases in forebrain glutathione (GSH), and increased levels of transcripts coding for proteins involved in GSH synthesis and detoxification pathways, as revealed by quantitative PCR analysis. Given the general importance of oxidative stress as a mediator of neurodegeneration we propose that an increase in brain L-cysteine levels could be protective in HD. Furthermore, cystamine was dramatically protective against 3-nitropropionic acid-induced striatal injury in mice. We suggest that cystamine's neuroprotective effect in HD transgenic mice results from pleiotropic effects that include transglutaminase inhibition and antioxidant activity.

The proteome is the protein compliment of the genome and is the result of genetic expression, ribosomal synthesis and proteolytic degradation. Proteins participate in most major cell processes and their function is highly regulated by post-translational modifications such as phosphorylation and glycosylation. As a result, neurotoxicant-induced changes in protein levels, function or regulation could have a negative impact on neuronal viability. At the molecular level, direct oxidative or covalent modifications of individual proteins by various chemicals or drugs is likely to lead to perturbation of tertiary structure and a loss of function. The proteome and the functional determinants of its individual protein components are, therefore, likely targets of neurotoxicant action and resulting characteristic disruptions could be critically involved in corresponding mechanisms of neurotoxicity. Clearly, investigating changes in the proteome can provide important clues for deciphering mechanisms of toxicant action and, therefore, proteomics, the study of the proteome, is currently, and will likely remain, a significant experimental approach for mechanistic research in neurotoxicology. The purpose of this review is to discuss proteomics as a tool for neurotoxicological investigations. A variety of classic proteomic techniques (e.g. liquid chromatography (LC)/tandem mass spectroscopy, two-dimensional gel image analysis) as well as more recently developed approaches (e.g. two-hybrid systems, antibody arrays, protein chips, isotope-coded affinity tags, ICAT) are available to determine protein levels, identify components of multiprotein complexes and to detect post-translational changes. Proteomics, therefore, offers a comprehensive overview of cell proteins, and in the case of neurotoxicant exposure, can provide quantitative data regarding changes in corresponding expression levels and/or post-translational modifications that might be associated with neuron injury.

17alpha-ethinylestradiol (EE2), used for birth control in humans, is a potent estrogen that is found in wastewater at low concentrations (ng/L). EE2 has the ability to interfere with the endocrine system of fish, affecting reproduction which can result in population level effects. The objective of this study was to determine if dietary exposure to EE2 would alter gene expression patterns and key pathways in the liver and ovary and whether these could be associated with reproductive endpoints in female largemouth bass during egg development. Female LMB received 70 ng EE2/g feed (feed administered at 1% of body weight) for 60 days. EE2 dietary exposure significantly reduced plasma vitellogenin concentrations by 70%. Hepatosomatic and gonadosomatic indices were also decreased with EE2 feeding by 38.5% and 40%, respectively. Transcriptomic profiling revealed that there were more changes in steady state mRNA levels in the liver compared to the ovary. Genes associated with reproduction were differentially expressed such as vitellogenin in the liver and aromatase in the gonad. In addition, a set of genes related with oxidative stress (e.g. glutathione reductase and glutathione peroxidase) were identified as altered in the liver and genes associated with the immune system (e.g. complement component 1, and macrophage-inducible C-type lectin) were altered in the gonad. In a follow-up study with 0.2 ng EE2/g feed for 60 days, similar phenotypic and gene expression changes were observed that support these findings with the higher concentrations. This study provides new insights into how dietary exposure to EE2 interferes with endocrine signaling pathways in female LMB during a critical period of reproductive oogenesis.

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.

Organophosphorus (OP) compounds have been reported to inhibit Ca/Mg-ATPase, but the relevance of this inhibition to organophosphate-induced delayed neuropathy (OPIDN) has not been explored. To determine if inhibition of this enzyme was related to the development of OPIDN, neuropathic and nonneuropathic OP compounds were sted for their ability to inhibit Ca-stimulated ATPase activity in the P2 synaptosomal fraction from hen brain. Following in vitro exposure to 10(-3) to 10(-5) M OP compounds, Ca-stimulated ATPase activity was inhibited by chlorpyrifos, chlorpyrifos-oxon, phenyl saligenin phosphate (PSP), and tri-o-tolyl phosphate (TOTP), but not by parathion, paraoxon, or diisopropyl fluorophosphate (DFP). Further investigation of inhibition induced by chlorpyrifos determined that inhibition was noncompetitive with respect to calcium and ATP. OP compound hydrophobicity was well correlated with in vitro inhibition of Ca-stimulated ATPase, suggesting that OP compounds interact with membrane lipids, and this interaction may contribute to the noncompetitive inhibition of Ca-stimulated ATPase that was observed. Subsequent to in vivo exposure, DFP, but not PSP, produced inhibition of Ca-stimulated ATPase activity in the hen brain P2 synaptosomal fraction. These data indicate that inhibition of Ca-stimulated ATPase activity is not correlated to neuropathic potential and demonstrate that inhibition of Ca/Mg-ATPase is not responsible for OPIDN.

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, P0.05) 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 (P0.05). 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.

The loss of epithelial barrier integrity in bronchial and bron- chiolar 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 99m Tc- 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-tetra- decane (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.

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.

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.

Acrylamide (ACR) is an electrophilic unsaturated carbonyl derivative that produces neurotoxicity by forming irreversible Michael-type adducts with nucleophilic sulfhydryl thiolate groups on cysteine residues of neuronal proteins. Identifying specific proteins targeted by ACR can lead to a better mechanistic understanding of the corresponding neurotoxicity. Therefore, in the present study, the ACR-adducted proteome in exposed primary immortalized mesencephalic dopaminergic cells (N27) was determined using tandem mass spectrometry (LTQ-Orbitrap). N27 cells were characterized based on the presumed involvement of CNS dopaminergic damage in ACR neurotoxicity. Shotgun proteomics identified a total of 15,243 peptides in N27 cells of which 103 unique peptides exhibited ACR-adducted Cys groups. These peptides were derived from 100 individual proteins and therefore ~0.7% of the N27 cell proteome was adducted. Proteins that contained ACR adducts on multiple peptides included annexin A1 and pleckstrin homology domain-containing family M member 1. Sub-network enrichment analyses indicated that ACR-adducted proteins were involved in processes associated with neuron toxicity, diabetes, inflammation, nerve degeneration and atherosclerosis. These results provide detailed information regarding the ACR-adducted proteome in a dopaminergic cell line. The catalog of affected proteins indicates the molecular sites of ACR action and the respective roles of these proteins in cellular processes can offer insight into the corresponding neurotoxic mechanism.

The pulmonary toxicity of a substance depends on a number of chemical and physical characteristics, including the solubility of the compounds. In the lung, insoluble forms of metals may be more tumorigenic than soluble forms despite the fact that this effect has not been quantitated and the mechanism of action has not been elucidated. The toxic effects of slightly soluble forms of As(III) and As(V) were evaluated by determining alteration in function of pulmonary alveolar macrophages (PAM) following in vivo and in vitro exposure. Male Sprague-Dawley rats were used throughout. Twenty-four hours following intratracheal instillation of 1 mg/kg (as arsenic) of either arsenic trisulfide (As(III)) or calcium arsenate (As(V)), PAM were lavaged and analyzed for alterations in superoxide (O-2(-)), and tumor necrosis factor (TNF-alpha) production. There were no differences in bronchoalveolar lavage fluid TNF-alpha. PAM. lavaged from As(V)-exposed animals showed significant increases in O-2(-) production and in basal release of TNF-alpha. PAM lavaged from animals receiving As(III) did not show significant alterations. To test the direct effects of arsenic, PAM were lavaged from control animals and exposed to concentrations of 0,1 to 300 mu g/ml arsenic in vitro for up to 24 hr. Doses used were not cytotoxic to PAM, since LDH release was not significantly increased. Significant dose-dependent inhibition of O-2(-) production was only evident after 24 hr exposure to arsenicals. Both As(III) and As(V) produced inhibition at concentrations of 10 mu g/ml. Suppression of LPS-induced release of TNF-alpha also occurred at similar concentrations for both arsenicals (4-5 mu g/ml). Neither arsenical inhibited prostaglandin E(2) production. Measurement of soluble arsenic concentrations indicated dissolution of the compounds could not account for all of the effects seen. Arsenic-induced alteration in PAM function may compromise host defense.

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: NH 2 ) 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 24 h 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 NH 2 (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 24 h 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.

Organochlorine pesticides (OCPs) such as dieldrin are a persistent class of aquatic pollutants that cause adverse neurological and reproductive effects in vertebrates. In this study, female and male largemouth bass (Micropterus salmoides) (LMB) were exposed to 3mg dieldrin/kg feed in a 2 month feeding exposure (August-October) to (1) determine if the hypothalamic transcript responses to dieldrin were conserved between the sexes; (2) characterize cell signaling cascades underlying dieldrin neurotoxicity; and (3) determine whether or not co-feeding with 17β-estradiol (E(2)), a hormone with neuroprotective roles, mitigates responses in males to dieldrin. Despite also being a weak estrogen, dieldrin treatments did not elicit changes in reproductive endpoints (e.g. gonadosomatic index, vitellogenin, or plasma E(2)). Sub-network (SNEA) and gene set enrichment analysis (GSEA) revealed that neuro-hormone networks, neurotransmitter and nuclear receptor signaling, and the activin signaling network were altered by dieldrin exposure. Most striking was that the majority of cell pathways identified by the gene set enrichment were significantly increased in females while the majority of cell pathways were significantly decreased in males fed dieldrin. These data suggest that (1) there are sexually dimorphic responses in the teleost hypothalamus; (2) neurotransmitter systems are a target of dieldrin at the transcriptomics level; and (3) males co-fed dieldrin and E(2) had the fewest numbers of genes and cell pathways altered in the hypothalamus, suggesting that E(2) may mitigate the effects of dieldrin in the central nervous system.

Ethanol exposure causes neurotoxicity, where neuroinflammation has been proposed to contribute to ethanol neurotoxicity. In addition to astroglia, microglia, as resident immune cells in the central nervous system, have been implicated as a key contributor to the neuroimmune and inflammatory processes. However, little is known regarding the role of microglia in alcohol-induced neuronal dysfunction. In this chapter, we describe a method that provides an effective and unbiased global-scale analysis for relative quantitation of protein expression in microglial cells to elucidate the molecular mechanisms underlying microglial activation after ethanol exposure. The approach involves stable isotope labeling with amino acids in cell culture followed by mass spectrometric analysis of stable isotope-labeled proteins derived from cultured microglial cells and represents a powerful tool that can be used for general assessment of microglial response at the protein level.

Acrylamide (ACR) intoxication is associated with selective nerve terminal damage in the central and peripheral nervous systems. As a soft electrophile, ACR could form adducts with nucleophilic sulfhydryl groups on cysteine residues of kelch-like erythroid cell-derived protein with CNS homology-associated protein 1 (Keap1) leading to dissociation of the transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2 activation of the antioxidant-responsive element (ARE) and subsequent upregulated gene expression of phase II detoxification enzymes and anitoxidant proteins might provide protection in neuronal regions with transcriptional capabilities (e.g., cell body). In contrast, non-transcriptional cell regions (axons, nerve terminals) might be vulnerable to electrophile-induced damage. To test this possibility, immunoblot analysis was used to measure protein products of Nrf2-activated ARE genes in nerve terminals and in cytosolic/nuclear factions of neuronal cell bodies isolated from rats intoxicated at two different ACR dose-rates; i.e., 50 mg/kg/d × 10 days, 21 mg/kg/d × 38 days. To detect possible differences in cell-specific induction, the cytoprotective response to ACR intoxication was determined in hepatic cells. Results show that control brain and hepatic cell fractions exhibited distinct subcellular distributions of Nrf2, Keap1 and several ARE protein products. ACR intoxication, however, did not alter the levels of these proteins in synaptosomal, brain cytoplasm or liver cell fractions. These data indicate that ACR was an insufficient electrophilic signal for ARE induction in all subcellular fractions tested. Because a cytoprotective response was not induced in any fraction, nerve terminal vulnerability to ACR cannot be ascribed to the absence of transcription-based defense mechanisms in this neuronal region.

In freshwater fish, aluminum is a well-recognized gill toxicant, although responses are influenced by pH. Aluminum nanomaterials are being used in diverse applications that are likely to lead to environmental release and exposure. However, it is unclear if the effects of nanoparticulate aluminum are similar to those of other forms of aluminum or require special consideration. To examine the acute toxicological effects of exposure to aluminum nanoparticle (Al-NP)s, adult female zebrafish were exposed to either Al-NPs or aluminum chloride for up to 48 hours in moderately hard fresh water. Al-NPs introduced into test water rapidly aggregated and up to 80% sedimented from the water column during exposures. No mortality was caused by concentrations of Al-NP up to 12.5 mg/L. After exposure, tissue concentrations of aluminum, effects on gill morphology, Na+, K+ -ATPase (NKA) activity, and global gene expression patterns were examined. Exposure to both aluminum chloride and nanoparticulate aluminum resulted in a concentration dependent decrease in sodium potassium ATPase activity, although Al-NP exposure did not alter gill morphology as measured by filament widths. Decreased ATPase activity coincided with decreases in filamental NKA staining and mucous cell counts. Analysis of gill transcriptional responses demonstrated that exposure to 5 mg/L Al-NP only resulted in significant changes in expression of two genes, whereas aluminum chloride exposure significantly affected the expression of 105 genes. Taken together, these results indicate that nanoparticulate aluminum has little acute toxicity for zebrafish in moderately hard freshwater.

Dieldrin is a persistent organochlorine pesticide that induces neurotoxicity in the vertebrate central nervous system and impairs reproductive processes in fish. This study examined the molecular events produced by subchronic dietary exposures to 2.95 mg dieldrin/kg feed in the neuroendocrine brain of largemouth bass, an apex predator. Microarrays, proteomics, and pathway analysis were performed to identify genes, proteins, and cell processes altered in the male hypothalamus. Fifty-four genes were induced, and 220 genes were reduced in steady-state levels (p < 0.001; fold change greater than ± 1.5). Functional enrichment analysis revealed that the biological gene ontology categories of stress response, nucleotide base excision repair, response to toxin, and metabolic processes were significantly impacted by dieldrin. Using isobaric tagging for relative and absolute quantitation, 90 proteins in the male hypothalamus were statistically evaluated for changes in protein abundance. Several proteins altered by dieldrin are known to be associated with human neurodegenerative diseases, including apolipoprotein E, microtubule-associated tau protein, enolase 1, stathmin 1a, myelin basic protein, and parvalbumin. Proteins altered by dieldrin were involved in oxidative phosphorylation, differentiation, proliferation, and cell survival. This study demonstrates that a subchronic exposure to dieldrin alters the abundance of messenger RNAs and proteins in the hypothalamus that are associated with cell metabolism, cell stability and integrity, stress, and DNA repair.

Acrolein and 4-hydroxy-2-nonenal (HNE) are byproducts of lipid peroxidation and are thought to play central roles in various traumatic injuries and disease states that involve cellular oxidative stress, for example, spinal cord trauma, diabetes, and Alzheimer's disease. In this review, we will discuss the chemical attributes of acrolein and HNE that determine their toxicities. Specifically, these aldehydes are classified as type 2 alkenes and are characterized by an alpha,beta-unsaturated carbonyl structure. This structure is a conjugated system that contains mobile pi-electrons. The carbonyl oxygen atom is electronegative and can promote the withdrawal of mobile electron density from the beta-carbon atom causing regional electron deficiency. On the basis of this type of electron polarizability, both acrolein and HNE are considered to be soft electrophiles that preferentially form 1,4-Michael type adducts with soft nucleophiles. Proteomic, quantum mechanical, and kinetic data will be presented, indicating that cysteine sulfhydryl groups are the primary soft nucleophilic targets of acrolein and HNE. This is in contrast to nitrogen groups on harder biological nucleophiles such as lysine or histidine residues. The toxicological outcome of adduct formation is not only dependent upon residue selectivity but also the importance of the targeted amino acid in protein function or structure. In attempting to discern the toxicological significance of a given adduct, we will consider the normal roles of cysteine, lysine, and histidine residues in proteins and the relative merits of corresponding adducts in the manifestations of diseases or toxic states. Understanding the molecular actions of acrolein and HNE could provide insight into many pathogenic conditions that involve initial cellular oxidative stress and could, thereby, offer new efficacious avenues of pharmacological defense.

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 1 mg/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.

Acrylamide (ACR) is a conjugated type-2 alkene that produces synaptic toxicity presumably by sulfhydryl adduction. The alpha,beta-unsaturated carbonyl of ACR is a soft electrophile and, therefore, adduction of nucleophilic thiol groups could occur through a conjugate (Michael) addition reaction. To address the mechanism of thiol adduct formation and corresponding neurotoxicological importance, we defined structure-toxicity relationships among a series of conjugated type-2 alkenes (1 microM-10mM), which included acrolein and methylvinyl ketone. Results show that exposure of rat striatal synaptosomes to these chemicals produced parallel, concentration-dependent neurotoxic effects that were correlated to loss of free sulfhydryl groups. Although differences in relative potency were evident, all conjugated analogs tested were equiefficacious with respect to maximal neurotoxicity achieved. In contrast, nonconjugated alkene or aldehyde congeners did not cause synaptosomal dysfunction or sulfhydryl loss. Acrolein and other alpha,beta-unsaturated carbonyls are bifunctional (electrophilic reactivity at the C-1 and C-3 positions) and could produce in vitro neurotoxicity by forming protein cross-links rather than thiol monoadducts. Immunoblot analysis detected slower migrating, presumably derivatized, synaptosomal proteins only at very high acrolein concentrations (>or= 25 mM). Exposure of synaptosomes to high concentrations of ACR (1M), N-ethylmaleimide (10mM), and methyl vinyl ketone (MVK) (100mM) did not alter the gel migration of synaptosomal proteins. Furthermore, hydralazine (1mM), which blocks the formation of protein cross-links, did not affect in vitro acrolein neurotoxicity. Thus, type-2-conjugated alkenes produced synaptosomal toxicity that was linked to a loss of thiol content. This is consistent with our hypothesis that the mechanism of ACR neurotoxicity involves formation of Michael adducts with protein sulfhydryl groups.

Largemouth bass (LMB) in Central Florida living on sites with high levels of organochlorine pesticides (OCPs) have exhibited poor reproductive success and altered steroid profiles. The mechanism underlying these changes is unknown, however changes in the rate of steroid metabolism could alter steroid homeostasis. Members of the CYP2 and CYP3A families play a significant role in the metabolism of many xenobiotics and endogenous compounds, including sex steroids. Therefore, the goal of this study was to identify members of the CYP2 and CYP3A families in LMB and characterize the effects of OCP exposure on their expression. Full-length clones of two CYP3A isoforms were obtained from LMB liver, CYP3A68 and 3A69, which exhibited significant sequence divergence. Full-length clones for CYP2N14 and CYP2P11 were also obtained from LMB liver. Steady-state mRNA levels of each of these CYPs increased in both sexes between early reproductive phase (December) and peak reproductive phase (March). Expression of CYP3A68 and CYP2P11 was sexually dimorphic during peak reproductive phase with 2-fold higher expression in females and males, respectively. Foodborne exposure to 46 ppm p,p′-DDE or 0.8 ppm dieldrin for 30 days did not have a significant effect on expression of CYPs. However, 4 months exposure to p,p′-DDE induced CYP3A68 and 3A69 expression in both sexes, while dieldrin produced weak induction of CYP3A68 and suppressed CYP3A69 expression in females, but had no effect on males. Neither p,p′-DDE nor dieldrin significantly altered the expression of CYP2P11 or CYP2N14. This work demonstrates that there are significant changes in CYP expression that occur during LMB reproduction which can be modified by exposure to OCPs.

Many structurally diverse chemicals (e.g., acrylamide, 2,4-dithiobiuret, methylmercury) are electrophiles and cause synaptic dysfunction by unknown mechanisms. The purpose of this Forum review is to discuss the possibility that highly nucleophilic cysteine thiolate groups within catalytic triads of synaptic proteins represent specific and necessary targets for electrophilic neurotoxicants. Most of these toxicants share the ability to adduct or otherwise modify nucleophilic sulfhydryl groups. It is also now recognized that synaptic activity is regulated by the redox state of certain cysteine sulfhydryl groups on proteins. Electrophilic neurotoxicants might, therefore, produce synaptic toxicity by modifying these thiols. Because most proteins contain cysteine residues, target specificity is an issue that significantly detracts from the mechanistic validity of this hypothesis. However, recent research indicates that these thiolates are receptors for the endogenous nitric oxide (NO) pathway and that subsequent reversible S-nitrosylation finely regulates a broad spectrum of synaptic activities. We hypothesize that electrophilic neurotoxicants selectively adduct/derivatize NO-receptor thiolates in catalytic triads and that the resulting loss of fine gain control impairs neurotransmission and produces neurotoxicity. This proposal has mechanistic implications for a large class of electrophilic chemicals used in the agricultural and industrial sectors. In addition, research based on this hypothesis could provide mechanistic insight into neurodegenerative conditions such as Parkinsonism and Alzheimer's disease that presumably involve endogenous production of neurotoxic electrophiles (e.g., acrolein, 4-hydroxy-2-nonenal). The proposed mechanism of electrophilic neurotoxicants represents a new and exciting experimental framework for mechanistic research in human neuropathological conditions associated with toxicant exposure or disease-based processes.

Dieldrin and p,p'-DDE are ubiquitous contaminants known to act as endocrine disruptors, causing impaired development and reproduction in fish and wildlife. In order to elucidate the mechanisms by which dieldrin and p,p'-DDE cause endocrine disruption in largemouth bass (Micropterus salmoides), fish were exposed subchronically through the diet to both contaminants. Following 120 days of exposure, p,p'-DDE decreased estradiol in females, but increased 11-ketotestosterone in both sexes. Dieldrin on the other hand, decreased estradiol and 11-ketotestosterone in both sexes. Both pesticides also altered steady state mRNA expression levels of a set of genes chosen to represent three possible mechanisms of endocrine disruption: (1) direct interaction with soluble sex steroid receptors, (2) biosynthesis of endogenous sex hormones, and (3) metabolism of endogenous hormones. p,p'-DDE acted as a weak estrogen, increasing the expression of vitellogenin and estrogen receptor alpha in the liver. p,p'-DDE also altered the expression of genes involved in the synthesis of endogenous hormones as well as their metabolism. Dieldrin, on the other hand, only altered expression of vitellogenin and not estrogen receptor alpha. Dieldrin also altered the expression of genes involved in hormone synthesis and metabolism, and it dramatically lowered plasma hormone levels. Both pesticides targeted expression of genes involved in all three modes of action, suggesting that they each have multiple modes of action.

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.

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.

The mechanism of arsine (AsH 3 ) toxicity is not completely understood. In this investigation, we determined AsH 3 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 AsH 3 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 AsH 3 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 AsH 3 . At 24 h, both arsenic species showed similar toxicity. In renal cortical epithelial cells, AsH 3 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 AsH 3 produced toxicity in tissues, in addition to blood, and that toxicity of arsenicals is arsenic species- and tissue-dependent.

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- isor =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 ator =20 microM Hg, except albumin, which protected ator =20 microM Hg. In cysteine-free but cystine-containing medium, two 1:1 Hg(II):thiol complexes were toxic ator =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.

The ability of bromine and rat liver microsomes (RLM) to convert organophosphorus (OP) protoxicants to esterase inhibitors was determined by measuring acetylcholinesterase (AChE) and neuropathy target esterase (NTE) inhibition. Species specific differences in susceptibility to esterase inhibition were determined by comparing the extent of esterase inhibition observed in human neuroblastoma cells and hen, bovine, and rodent brain homogenates. OP protoxicants examined included tri-o-tolyl phosphate (TOTP), O-ethyl O-p-nitrophenyl phenylphosphonothioate (EPN), leptophos, fenitrothion, fenthion, and malathion. Bromine activation resulted in greater AChE inhibition than that produced by RLM activation for equivalent concentrations of fenitrothion, malathion, and EPN. For EPN and leptophos, bromine activation resulted in greater inhibition of NTE than RLM. Only preincubation with RLM activated TOTP; resultant inhibition of AChE was less in hen brain (13 +/- 3%) than in neuroblastoma cells (73 +/- 1%) at 10(-6) M. In contrast, 10(-6) M RLM-activated TOTP produced more inhibition of hen brain NTE (89 +/- 6%) than NTE of human neuroblastoma cells (72 +/- 7%). Human neuroblastoma cells and brain homogenates from hens, the accepted animal model for study of OP-induced neurotoxicity, were relatively similar in sensitivity to esterase inhibition. Homogenates from hens were more sensitive to NTE inhibition induced by phenyl saligenin phosphate (PSP), an active congener of TOTP, than were homogenates from less susceptible species (mouse, rat, bovine). AChE of hen brain homogenates was also more sensitive than homogenates from other species to malaoxon, the active form of malathion.

In order to perform in vitro testing of esterase inhibition caused by organophosphorous (OP) protoxicants, simple, reliable methods are needed to convert protoxicants to their esterase-inhibiting forms. Incubation of parathion or chlorpyrifos with 0.05% bromine solution or uninduced rat liver microsomes (RLM) resulted in production of the corresponding oxygen analogs of these OP compounds and markedly increased esterase inhibition in SH-SY5Y human neuroblastoma cells. Neither activation system affected cell viability or the activity of AChE or NTE in the absence of OP compounds. Although parathion and chlorpyrifos were activated by RLM, bromine activation required fewer steps and produced more esterase inhibition for a given concentration of chlorpyrifos. However, RLM activation of OP protoxicants produced metabolites other than oxygen analogs and may, therefore, be more relevant as a surrogate for OP biotransformation in vivo. This methodology makes the use of intact cells for in vitro testing of esterase inhibition caused by protoxicant organophosphate compounds a viable alternative to in vivo tests.