Steroidogenic acute regulatory protein transcription is regulated by estrogen receptor signaling in largemouth bass ovary.

Estrogenic contaminants in the environment are linked to the occurrence of reproductive abnormalities in many aquatic species, including largemouth bass (Micropterus salmoides; LMB). Previous work has shown that many different types of xenoestrogens regulate expression of the Steroidogenic Acute Regulatory protein (StAR), a cholesterol-transporting protein vital to steroid hormone biosynthesis; however, the regulatory mechanisms of StAR are incompletely characterized in fish. To learn more about endogenous expression patterns of StAR in the ovary, LMB were collected from the St. John's River (Florida, USA) over an entire breeding season to investigate StAR expression. Plasma 17ß-estradiol (E2) and StAR mRNA levels were positively correlated in females, and StAR mRNA levels displayed ~ 100-fold increase between primary oocyte growth stages and final maturation. To further study the regulation of StAR, female LMB in the laboratory were fed at ≃2% of their weight on a diet laden with 17α-ethinylestradiol (EE2, 70 or 200 ng EE2 per gram feed). Diets were designed to achieve a physiologically-relevant exposure to EE2, and StAR expression was assessed in vivo. We observed a dose-dependent suppression of StAR mRNA levels, however both diets led to high, pharmacological levels in the blood and do not represent normal physiological ranges of estrogens. In the 200 ng EE2/gm feed group, ovarian StAR mRNA levels were suppressed to approximately 5% of that of the LMB control group. These investigations suggest that LMB StAR increases in expression during oocyte maturation and that it is suppressed by E2 feedback when estrogen levels are high, through the HPG axis. A 2.9 kb segment of the LMB StAR promoter was examined for putative E2 response elements using in silico software, and a putative estrogen receptor binding element (ERE/-1745) was predicted in the promoter. The functionality of the ERE was examined using MA-10 mouse Leydig cells transfected with the LMB StAR promoter. Estrogen receptor (ER) interaction with ERE/-1745 was evaluated under basal and human chorionic gonadotropin (hCG)-treated conditions in the presence and absence of E2. Chromatin immunoprecipitation (ChIP) experiments revealed that ESR1 binding to the promoter was enriched under basal conditions and E2 exposure elicited an increase in enrichment (4-fold) above that observed under basal conditions. ESR2 was not strongly enriched at the ERE/-1745 site, suggesting that StAR may be preferentially regulated by LMB estrogen receptor 1 (esr1). Taken together, these different experiments provide evidence that LMB StAR is under the control of estrogens and that ESR1 binds directly to the LMB StAR promoter in an E2-responsive manner.

Proteomic analysis of rat microglia establishes a high-confidence reference data set of over 3000 proteins.

Highly aggressively proliferating immortalized (HAPI) microglial cells have been used as an in vitro model for investigating key microglial functions including inflammatory, neurotoxic, and phagocytic activities. Through the use of offline strong cation-exchange fractionation followed by inline reversed-phase chromatographic separation and tandem mass spectrometric analysis on a hybrid linear ion trap-Orbitrap instrument, the HAPI microglial proteome was characterized to a depth of 3006 unique protein groups. Upon bioinformatic analysis of the HAPI proteome data set, enrichment was observed for processes relevant to microglial function including those associated with immune system response. This study marks the most comprehensive reference data set generated to date for the rat microglial proteome.

Involvement of dopaminergic neuronal cystatin C in neuronal injury-induced microglial activation and neurotoxicity.

Factors released from injured dopaminergic (DA) neurons may trigger microglial activation and set in motion a vicious cycle of neuronal injury and inflammation that fuels progressive DA neurodegeneration in Parkinson's disease. In this study, using proteomic and immunoblotting analysis, we detected elevated levels of cystatin C in conditioned media (CM) from 1-methyl-4-phenylpyridinium and dieldrin-injured rat DA neuronal cells. Immunodepletion of cystatin C significantly reduced the ability of DA neuronal CM to induce activation of rat microglial cells as determined by up-regulation of inducible nitric oxide synthase, production of free radicals and release of proinflammatory cytokines as well as activated microglia-mediated DA neurotoxicity. Treatment of the cystatin C-containing CM with enzymes that remove O- and sialic acid-, but not N-linked carbohydrate moieties markedly reduced the ability of the DA neuronal CM to activate microglia. Taken together, these results suggest that DA neuronal cystatin C plays a role in the neuronal injury-induced microglial activation and neurotoxicity. These findings from the rat DA neuron-microglia in vitro model may help guide continued investigation to define the precise role of cystatin C in the complex interplay among neurons and glia in the pathogenesis of Parkinson's disease.

Application of the Hard and Soft, Acids and Bases (HSAB) theory to toxicant--target interactions.

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.

ß-dicarbonyl enolates: a new class of neuroprotectants.

Curcumin, phloretin and structurally related phytopolyphenols have well-described neuroprotective properties that appear to be at least partially mediated by 1,3-dicarbonyl enol substructures that form nucleophilic enolates. Based on their structural similarities, we tested the hypothesis that enolates of simple 1,3-dicarbonyl compounds such as acetylacetone might also possess neuroprotective actions. Our results show that the ß-diketones, particularly 2-acetylcyclopentanone, protected rat striatal synaptosomes and a neuronal cell line from thiol loss and toxicity induced by acrolein, an electrophilic α,ß-unsaturated aldehyde. The 1,3-dicarbonyl compounds also provided substantial cytoprotection against toxicity induced by hydrogen peroxide in a cellular model of oxidative stress. Initial chemical characterization in cell-free systems indicated that the 1,3-dicarbonyl compounds acted as surrogate nucleophilic targets that slowed the rate of sulfhydryl loss caused by acrolein. Although the selected 1,3-dicarbonyl congeners did not scavenge free radicals, metal ion chelation was a significant property of both acetylacetone and 2-acetylcyclopentanone. Our data suggest that the 1,3-dicarbonyl enols represent a new class of neuroprotectants that scavenge electrophilic metal ions and unsaturated aldehydes through their nucleophilic enolate forms. As such, these enols might be rational candidates for treatment of acute or chronic neurodegenerative conditions that have oxidative stress as a common molecular etiology.

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus.

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.

Molecular mechanism of glyceraldehyde-3-phosphate dehydrogenase inactivation by α,ß-unsaturated carbonyl derivatives.

α,ß-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,ß-unsaturated carbonyl toxicants inhibited GAPDH activity in a concentration- and time-dependent manner. The rank order of enzyme inhibition (K(I)) (i.e., ACR ≪ MVK < acrolein) 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,ß-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.

Cloning and expression of the translocator protein (18 kDa), voltage-dependent anion channel, and diazepam binding inhibitor in the gonad of largemouth bass (Micropterus salmoides) across the reproductive cycle.

Cholesterol transport across the mitochondrial membrane is rate-limiting for steroidogenesis in vertebrates. Previous studies in fish have characterized expression of the steroidogenic acute regulatory protein, however the function and regulation of other genes and proteins involved in piscine cholesterol transport have not been evaluated. In the current study, mRNA sequences of the 18 kDa translocator protein (tspo; formerly peripheral benzodiazepine receptor), voltage-dependent anion channel (vdac), and diazepam binding inhibitor (dbi; also acyl-CoA binding protein) were cloned from largemouth bass. Gonadal expression was examined across reproductive stages to determine if expression is correlated with changes in steroid levels and with indicators of reproductive maturation. In testis, transcript abundance of tspo and dbi increased with reproductive maturation (6- and 23-fold maximal increase, respectively) and expression of tspo and dbi was positively correlated with reproductive stage, gonadosomatic index (GSI), and circulating levels of testosterone. Testis vdac expression was positively correlated with reproductive stage and GSI. In females, gonadal tspo and vdac expression was negatively correlated with GSI and levels of plasma testosterone and 17ß-estradiol. Ovarian dbi expression was not correlated with indicators of reproductive maturation. These studies represent the first investigation of the steroidogenic role of tspo, vdac, and dbi in fish. Findings suggest that cholesterol transport in largemouth bass testis, but not in ovary, may be transcriptionally-regulated, however further investigation will be necessary to fully elucidate the role of these genes in largemouth bass steroidogenesis.

Investigation of acute nanoparticulate aluminum toxicity in zebrafish.

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.

Transcriptome and physiological effects of toxaphene on the liver-gonad reproductive axis in male and female largemouth bass (Micropterus salmoides).

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 17ß-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.

Molecular mechanisms of 4-hydroxy-2-nonenal and acrolein toxicity: nucleophilic targets and adduct formation.

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.

Type-2 alkenes mediate synaptotoxicity in neurodegenerative diseases.

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.

Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms.

Metallic nanoparticles are among the most widely used types of engineered nanomaterials; however, little is known about their environmental fate and effects. To assess potential environmental effects of engineered nanometals, it is important to determine which species are sensitive to adverse effects of various nanomaterials. In the present study, zebrafish, daphnids, and an algal species were used as models of various trophic levels and feeding strategies. To understand whether observed effects are caused by dissolution, particles were characterized before testing, and particle concentration and dissolution were determined during exposures. Organisms were exposed to silver, copper, aluminum, nickel, and cobalt as both nanoparticles and soluble salts as well as to titanium dioxide nanoparticles. Our results indicate that nanosilver and nanocopper cause toxicity in all organisms tested, with 48-h median lethal concentrations as low as 40 and 60 microg/L, respectively, in Daphnia pulex adults, whereas titanium dioxide did not cause toxicity in any of the tests. Susceptibility to nanometal toxicity differed among species, with filter-feeding invertebrates being markedly more susceptible to nanometal exposure compared with larger organisms (i.e., zebrafish). The role of dissolution in observed toxicity also varied, being minor for silver and copper but, apparently, accounting for most of the toxicity with nickel. Nanoparticulate forms of metals were less toxic than soluble forms based on mass added, but other dose metrics should be developed to accurately assess concentration-response relationships for nanoparticle exposures.

Construction of a robust microarray from a non-model species (largemouth bass) using pyrosequencing technology.

A novel custom microarray for largemouth bass (Micropterus salmoides) was designed with sequences obtained from a normalized cDNA library using the 454 Life Sciences GS-20 pyrosequencer. This approach yielded in excess of 58 million bases of high-quality sequence. The sequence information was combined with 2,616 reads obtained by traditional suppressive subtractive hybridizations to derive a total of 31,391 unique sequences. Annotation and coding sequences were predicted for these transcripts where possible. 16,350 annotated transcripts were selected as target sequences for the design of the custom largemouth bass oligonucleotide microarray. The microarray was validated by examining the transcriptomic response in male largemouth bass exposed to 17beta-oestradiol. Transcriptomic responses were assessed in liver and gonad, and indicated gene expression profiles typical of exposure to oestradiol. The results demonstrate the potential to rapidly create the tools necessary to assess large scale transcriptional responses in non-model species, paving the way for expanded impact of toxicogenomics in ecotoxicology.

Proteomic analysis of rat striatal synaptosomes during acrylamide intoxication at a low dose rate.

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 x 7, 14, or 21 days) and their age-matched controls. The synaptosomal proteins of each experimental group were labeled with either light (12C9--control) or heavy (13C9--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.

Structure-toxicity analysis of type-2 alkenes: in vitro neurotoxicity.

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.

Fish 'n' chips: the use of microarrays for aquatic toxicology.

Gene expression analysis is changing the way that we look at toxicity, allowing toxicologists to perform parallel analyses of entire transcriptomes. While this technology is not as advanced in aquatic toxicology as it is for mammalian models, it has shown promise for determining modes of action, identifying biomarkers and developing "signatures" of chemicals that can be used for field and mixture studies. A major hurdle for the use of microarrays in aquatic toxicology is the lack of sequence information for non-model species. Custom arrays based on gene libraries enriched for genes that are expressed in response to specific contaminants have been used with excellent success for some non-model species, suggesting that this approach will work well for ecotoxicology and spurring on the sequencing of cDNA libraries for species of interest. New sequencing technology and development of repositories for gene expression data will accelerate the use of microarrays in aquatic toxicology. Notwithstanding the preliminary successes that have been achieved even with partial cDNA libraries printed on arrays, ecological samples present elevated challenges for this technology due to the high degree of variation of the samples. Furthermore, recent studies that show nonlinear toxic responses for ecological species underscore the necessity of establishing time and dose dependence of effects on gene expression and comparing these results with traditional markers of toxicity. To realize the full potential of microarrays, researchers must do the experiments required to bridge the gap between the 'omics' technologies and traditional toxicology to demonstrate that microarrays have predictive value in ecotoxicology.

Exposure to p,p'-DDE or dieldrin during the reproductive season alters hepatic CYP expression in largemouth bass (Micropterus salmoides).

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.

Several glutathione S-transferase isozymes that protect against oxidative injury are expressed in human liver mitochondria.

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, delta(5)-androstene-3,17-dione (ADI), and cumene hydroperoxide (CuOOH). Interestingly, the specific mitochondrial GST activities toward 4HNE, a highly toxic alpha,beta-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.

Synaptic cysteine sulfhydryl groups as targets of electrophilic neurotoxicants.

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.