Intersex related gene expression profiles in clams Scrobicularia plana: Molecular markers and environmental application.

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction.

Widespread contamination of coastal sediments in the Transmanche Channel with anti-androgenic compounds.

This study analysed the levels of androgen receptor antagonist activity in extracts of coastal sediments sampled from estuaries in southern UK and northern France. Anti-androgenic (AA) activity varied between <0.2 and 224.3±38.4µg flutamide equivalents/g dry weight of sediment and was significantly correlated with the total organic carbon and silt content of samples. AA activity was detected in tissues extracts of clams, Scrobicularia plana, sampled from a contaminated estuary, some of which was due to uptake of a series of 4 or 5 ring polycyclic aromatic hydrocarbons (PAHs). Initial studies also indicated that fractionated extracts of male, but not female, clams also contained androgen receptor agonist activity due to the presence of dihydrotestosterone in tissues. This study reveals widespread contamination of coastal sediments of the Transmanche region with anti-androgenic compounds and these contaminants should be investigated for their potential to disrupt sexual differentiation in aquatic organisms.

Intersex in Scrobicularia plana: transcriptomic analysis reveals novel genes involved in endocrine disruption.

Intersex, the appearance of female characteristics in male gonads, has been identified in a wide range of aquatic species worldwide, yet the underpinning molecular etiology remains uncharacterized. The presence of intersex has been shown to be a widespread phenomenon in bivalve, S. plana, populations from the southwest coast of the U.K., as well as inducible in an experimental exposure regime using endocrine disrupting compounds (EDCs). Herein, we use the suppressive subtractive hybridization approach to isolate differentially expressed transcripts in S. plana males exhibiting intersex. Transcripts involved in cell signaling, cell cycle control, energy production/metabolism, microtubule assembly, and sperm physiology are all highlighted as differentially expressed in intersex male clams. These provide both an insight into the molecular mechanisms of action involved in the development of intersex, as well as facilitating potential molecular-level "early warning" biomarkers of the condition.

Measurements of the genotoxic potential of (xeno-)oestrogens in the bivalve mollusc Scrobicularia plana, using the Comet assay.

There is increasing concern about the fate and effects of (geno)toxic and endocrine disrupting chemicals in sediments, highlighting the need to develop suitable monitoring tools. The deposit-feeding bivalve mollusc Scrobicularia plana has been put forward as a promising bioindicator of sediment contamination in estuaries. The recent demonstration of intersex in S. plana populations has been attributed to the feminisation of male clams following exposure to (xeno-)oestrogens, yet the mode of action of these endocrine disrupting chemicals (EDCs) remains largely unclear. One hypothesis that warrants further investigation is the possible involvement of genotoxicity. The first objective of this study was to assess whether the blood cells of S. plana are suitable for genotoxicity screening, using the alkaline single cell gel electrophoresis (Comet) assay. This was demonstrated successfully by exposing blood cells under in vitro conditions to a range of concentrations of the reference genotoxin hydrogen peroxide (H(2)O(2)): strong correlations between H(2)O(2) concentration and various comet parameters were found. Subsequently, the Comet assay was used to test whether the natural oestrogen 17beta-oestradiol (E2) and the synthetic (xeno)oestrogens ethinyloestradiol (EE2) and nonylphenol (NP) can produce genotoxic effects in S. plana, which might indicate possible involvement of mutagenicity in the mode of action of intersex development. In these short-term tests, clear genotoxic effects (significantly more DNA in the comet tail) were demonstrated by all EDCs, albeit only at high doses: 100 ng/L E2, 1 microg/L EE2 and 100 microg/L NP in vitro; and 1 microg/L E2 and 1mg/L NP after a 6-day in vivo exposure. Nevertheless, this study provides valuable preliminary data on the application and sensitivity of S. plana blood cells and suggests that the Comet assay is a useful tool, to screen for genotoxicity in in faunal clams and to examine further the links with higher order effects.

Application of biomarkers to assess the condition of European Marine Sites.

A series of European Marine Sites has been designated as Special Areas of Conservation (SAC) in England. The aim of this study was to develop a practical methodology to assess the condition of SACs by applying a suite of biomarkers. Biomarkers were applied to the blue mussel Mytilus edulis and the shore crab Carcinus maenas from the Fal and Helford SAC (Cornwall). Individual biomarkers provided useful diagnostic information on the activity of certain classes of contaminants and an integrated Biomarker Response Index (BRI) was used to achieve a more holistic understanding of the condition of the SAC. The BRI indicated that the general health of both organisms was impacted in the upper part of the SAC (Fal Estuary) which correlated well with known chemical hotspots and sources of contamination. The BRI allows a pragmatic way to prioritise SAC sites that may require further investigative studies.

Imposex levels and tributyltin pollution in Ria de Aveiro (NW Portugal) between 1997 and 2007: evaluation of legislation effectiveness.

Nucella lapillus imposex levels and organotin (OT) concentrations in water and female tissues were measured in samples collected from the Ria de Aveiro (NW Portugal) between 1997 and 2007. Vas deferens sequence index (VDSI), relative penis size index (RPSI), mean female penis length (FPL) and percentage of imposex affected females (%I) were used to determine imposex levels at each site. A significant temporal decline in imposex intensity was observed during the assessed period. Imposex decrease was evident after 2003 although improvements were most notable from 2005 to 2007, probably due to the implementation of the EU Council Regulation no.782/2003 forbidding further application of tributyltin (TBT) antifouling on vessels carrying EU flags. Despite these improvements, OT analysis in N. lapillus female tissues and water indicate there are still recent TBT inputs into the study area.

Pathologic modifications of alpha-synuclein in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated squirrel monkeys.

alpha-Synuclein expression is increased in dopaminergic neurons challenged by toxic insults. Here, we assessed whether this upregulation is accompanied by pathologic accumulation of alpha-synuclein and protein modifications (i.e. nitration, phosphorylation, and aggregation) that are typically observed in Parkinson disease and in other synucleinopathies. A single injection of the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to squirrel monkeys caused a buildup of alpha-synuclein but not of beta-synuclein or synaptophysin within nigral dopaminergic cell bodies. Immunohistochemistry and immunoelectron microscopy also revealed large numbers of dystrophic axons labeled with alpha-synuclein. Antibodies that recognize nitrated and phosphorylated (at serine 129) alpha-synuclein stained neuronal cell bodies and dystrophic axons in the midbrain of MPTP-treated animals. After toxicant exposure, alpha-synuclein deposition occurred at the level of neuronal axons in which amorphous protein aggregates were observed by immunoelectron microscopy. In a subset of these axons, immunoreactivity for alpha-synuclein was still evident after tissue digestion with proteinase K, further indicating the accumulation of insoluble protein. These data indicate that toxic injury can induce alpha-synuclein modifications that have been implicated in the pathogenesis of human synucleinopathies. The findings are also consistent with a pattern of evolution of alpha-synuclein pathology that may begin with the accumulation and aggregation of the protein within damaged axons.

The etiopathogenesis of Parkinson disease and suggestions for future research. Part II.

We are at a critical juncture in our knowledge of the etiology and pathogenesis of Parkinson disease (PD). It is clear that PD is not a single entity simply resulting from a dopaminergic deficit; rather it is most likely caused by a combination of genetic and environmental factors. Although there is extensive new information on the etiology and pathogenesis of PD, which may advance its treatment, new syntheses of this information are needed. The second part of this two-part, state-of-the-art review by leaders in PD research critically examines the research field to identify areas for which new knowledge and ideas might be helpful for treatment purposes. Topics reviewed in Part II are genetics, animal models, and oxidative stress.

Comparison of the neurotoxic effects of proteasomal inhibitors in primary mesencephalic cultures.

Impairment of the ubiquitin-proteasome system (UPS) has been implicated in the pathogenesis of Parkinson's disease (PD). Because the neurodegenerative process of PD results in a severe loss of dopaminergic cells, previous in vitro studies have investigated the possibility that these neurons may be particularly vulnerable to proteasomal inhibition. Results of this earlier work are difficult to compare, however, since they were obtained using different proteasomal inhibitors at various concentrations and under diverse culture conditions. Here, four UPS inhibitors, i.e., lactacystin, PSI, epoxomicin and MG-132, were directly evaluated in terms of their ability to damage dopaminergic and GABAergic neurons in primary rat mesencephalic cultures. Using a broad range of concentrations and different incubation lengths, we found that proteasomal inhibitors consistently killed both dopaminergic and GABAergic neurons. The degree of toxicity was slightly different, however, between the two neuronal populations. When measurements of neurotransmitter uptake were used as indicators of neuronal cell viability, the extent of reduction of dopamine uptake caused by proteasomal inhibitors was slightly greater than the decrease in GABA uptake. With PSI the difference in reduction of dopamine vs. GABA uptake was less than 10% and did not reach statistical significance. With the other three inhibitors, dopaminergic cells were up to 20% more affected than GABAergic neurons; this difference reached statistical significance only at specific concentrations and time points. Preincubation of cultures with alpha-methyl-p-tyrosine, an inhibitor of dopamine synthesis, reduced dopamine concentration by 65% but failed to significantly change lactacystin- and MG-132-induced damage to dopaminergic neurons. Data indicate a modest preferential toxicity of proteasomal inhibitors toward dopaminergic cells and thus only in part support the hypothesis that a selective vulnerability to UPS dysfunction underlies the pathogenesis of nigrostriatal degeneration in PD.

Alpha-synuclein expression in the substantia nigra of MPTP-lesioned non-human primates.

Changes in the expression of alpha-synuclein are likely to underlie its normal function as well as its role in pathological processes. The relationship between toxic injury and alpha-synuclein expression was assessed in the substantia nigra of squirrel monkeys treated with a single injection of MPTP and sacrificed 1 week or 1 month later. At 1 week, when stereological cell counting revealed only a small decrease (-10%) in the number of dopaminergic neurons, alpha-synuclein mRNA and protein were markedly enhanced. Increased alpha-synuclein immunoreactivity was evident at the level of neuronal fibers whereas nigral cell bodies were devoid of detectable protein. At 1 month post-MPTP, neuronal loss rose to 40%. Both alpha-synuclein mRNA and protein remained elevated but, noticeably, a robust alpha-synuclein immunoreactivity characterized a significant number of cell bodies. Neuromelanin granules are hallmarks of dopaminergic neurons in primates. Therefore, the number of alpha-synuclein-positive cells that also contained neuromelanin was counted throughout the substantia nigra. At 1 month, the vast majority of alpha-synuclein-immunoreactive neurons contained neuromelanin, and approximately 80% of the dopaminergic cell bodies that survived MPTP toxicity stained positive for alpha-synuclein. The results indicate that a single toxic insult is capable of inducing a sustained alpha-synuclein up-regulation in the primate brain. They support a direct relationship between neuronal injury and enhanced alpha-synuclein expression, and suggest that protein elevation within cell bodies may be a late feature of neurons that have endured a toxic stress.

Toxicity of redox cycling pesticides in primary mesencephalic cultures.

A loss of nigrostriatal dopaminergic neurons is the primary neurodegenerative feature of Parkinson's disease. Paraquat, a known redox cycling herbicide, has recently been shown to kill selectively nigrostriatal dopaminergic cells in the mouse model. The purpose of this study was to test the ability of paraquat and other redox cycling pesticides to damage dopaminergic neurons in primary mesencephalic cultures. Addition of paraquat, diquat, or benzyl viologen to mesencephalic cultures induced morphological changes (e.g., dystrophic neuronal processes) consistent with dopaminergic cell injury. The three pesticides also caused cell death as assessed by a reduction of the number of tyrosine hydroxylase-immunoreactive neurons and a dose-dependent decrease in [(3)H]dopamine uptake. Quite interestingly, diquat and benzyl viologen were significantly more toxic than paraquat, probably reflecting their more pronounced ability to trigger redox cycling reactions. The data support a role of redox cycling as a mechanism of dopaminergic cell degeneration and suggest that the property of redox cycling should be taken into consideration when evaluating putative environmental risk factors for Parkinson's disease.

Redox cycling of the herbicide paraquat in microglial cultures.

Mechanisms involved in paraquat neurotoxicity that selectively target nigrostriatal dopaminergic neurons remain relatively unknown. In this study, we tested the hypotheses that paraquat exposure leads to the production of reactive oxygen species (ROS) through a process of redox cycling and that microglia represent an important site for the initiation of redox cycling reactions. Addition of paraquat to N9 microglial cultures resulted in a dose- and time-dependent release of superoxide radicals. Other agents that share with paraquat the property of redox cycling, i.e., benzyl viologen and diquat, also induced a marked production of superoxide radicals by microglia. The ability of paraquat, benzyl viologen, and diquat to induce superoxide release was correlated to their one-electron reduction potentials and thus their tendency to redox cycle. Nitric oxide synthase and NADPH oxidase were identified as enzymatic sources of electrons that triggered paraquat redox cycling by microglia. Taken together, these data provide evidence in favor of a new mechanism by which microglia could play a role in oxidative injury during neurodegenerative processes. Microglial NOS and NADPH oxidase could promote the generation of ROS via the redox cycling of paraquat-like toxicants.

Levodopa in the treatment of Parkinson's disease: current controversies.

Levodopa is the most effective symptomatic agent in the treatment of Parkinson's disease (PD) and the "gold standard" against which new agents must be compared. However, there remain two areas of controversy: (1) whether levodopa is toxic, and (2) whether levodopa directly causes motor complications. Levodopa is toxic to cultured dopamine neurons, and this may be a problem in PD where there is evidence of oxidative stress in the nigra. However, there is little firm evidence to suggest that levodopa is toxic in vivo or in PD. Clinical trials have not clarified this situation. Levodopa is also associated with motor complications. Increasing evidence suggests that they are related, at least in part, to the short half-life of the drug (and its potential to induce pulsatile stimulation of dopamine receptors) rather than to specific properties of the molecule. Treatment strategies that provide more continuous stimulation of dopamine receptors provide reduced motor complications in MPTP monkeys and PD patients. These studies raise the possibility that more continuous and physiological delivery of levodopa might reduce the risk of motor complications. Clinical trials to test this hypothesis are underway. We review current evidence relating to these areas of controversy.

Aging of the nigrostriatal system in the squirrel monkey.

Increasing incidence of Parkinson's disease with advancing age suggests that age-related processes predispose the nigrostriatal dopaminergic system to neurodegeneration. Several hypotheses concerning the effects of aging on nigrostriatal neurons were assessed in this study using a non-human primate model. First, we examined the possibility that the total number of dopaminergic neurons decline in the substantia nigra as a function of age. Stereological counting based on both tyrosine hydroxylase immunoreactivity (TH-ir) and neuromelanin (NM) content revealed no difference in cell number between young, middle-aged and old squirrel monkeys. We then determined whether advancing age changed the relative proportion of neurons characterized by 1) TH-ir in the absence of NM, 2) the presence of both TH-ir and NM, or 3) NM without TH-ir. Indeed, a progressive age-related depletion of TH only cells was paralleled by an increase in NM only neurons. The possibility that these changes could underlie a functional impairment of the nigrostriatal system was supported by striatal dopamine measurements showing a decrease in older monkeys. Finally, we tested the hypotheses that aging may enhance cell vulnerability to injury and that different dopaminergic subpopulations display varying degrees of susceptibility. When monkeys were exposed to the neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, cell loss was markedly more pronounced in older animals, and the ranking of vulnerability was TH only < TH/NM < NM only cells. The data indicate that, even in the absence of an overall neuronal loss, changes in the characteristics of dopaminergic cells reflect functional deficits and increased vulnerability to injury with age. NM content appears to be an important marker of these age-related effects.