Jose Miguel Bras

Andrew Singleton

GENETIC SUSCEPTIBILITY IN PARKINSON'S DISEASE

It is hoped that an understanding of the genetic basis of

Parkinson's disease (PD) will lead to an appreciation of the molecular pathogenesis of disease, which in turn will highlight potential points of therapeutic intervention. It is also hoped that such an understanding will allow identification of individuals at risk for disease prior to the onset of motor symptoms. A large amount of work has already been performed in the identification of genetic risk factors for PD and some of this work, particularly those efforts that focus on genes implicated in monogenic forms of PD, have been successful, although hard won. A new era of gene discovery has begun, with the application of genome wide association studies; these promise to facilitate the identification of common genetic risk loci for complex genetic diseases. This is the first of several high throughput technologies that promise to shed light on the (likely) myriad genetic factors involved in this complex, late-onset neurodegenerative disorder.

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M A N U S C R I P T

INTRODUCTION

The exact pathobiologic basis for the progressive cellular and system dysfunction observed in Parkinson's disease (PD) has remained largely elusive since the recognition of this disorder as a distinct entity. While palliative treatments exist for this disease, it is hoped that understanding the molecular events that lead to PD will facilitate the creation of an etiologic based therapy that will halt or reverse the disease process. This hope is a primary motivation underlying work aimed at understanding the genetic basis of PD and related disorders.

Monogenic forms of parkinsonism with a clear Mendelian pattern of inheritance have been the most accessible in terms of the identification of genetic causes and contributors. This work has lead to the discovery of mutations in SNCA, PARK2, PINK1, PARK6 and LRRK2 as causes of primary parkinsonism and/or PD. These findings have been central to much of the investigation into the molecular underpinnings of PD. While the identification of these loci has been important, mutation of these genes is responsible for a relatively small proportion of most PD cases.

In parallel to work on monogenic PD a large amount of research has focused on identifying genetic variability that confers risk for, rather than causes, PD. This work aims not only to add insight into the molecular pathogenesis of PD, but also to create a risk profile for disease in the general population. For the most part risk variant identification is based on the tenet of the common-disease commonvariant hypothesis. This theory operates on the premise that common genetic

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variants underlie susceptibility for common diseases such as PD. The commondisease common-variant hypothesis is an idea that is the basis for the vast majority of genetic case control association studies and the impetus for initiatives such as the International Human Haplotype Map Project (www.hapmap.org).

There has been significant contention over the common disease common variant hypothesis with substantive support for the idea that rare mutations underlie the etiology of complex disorders. While these are often proposed as opposing theories they are not mutually exclusive; however, the current accessible technologies do not readily allow the investigation of rare mutations as a cause of common disease in a complete way.

In this review we will discuss the genetic risk loci discovered thus far in PD; we will limit our discussion to those loci for which a genetic variant or variants have been proposed, rather than extant loci for which no 'gene' has been identified.

We will extend our discussion to include a brief consideration of genome wide association studies in PD.

In general within the field of disease genetics the search for common genetic risk variants has been a difficult and mostly fruitless one. There are of course several reasons for this failure, particularly in a disease such as PD. Most prominent is the size of effect and thus the number of samples required to detect an effect.

Many studies were predicated on the idea that risk variants with effect sizes comparable to APO E ε4 in Alzheimer's disease may exist for PD; thus these

SNCA.

From an etiologic perspective this observation fits well with the discovery that multiplication of the SNCA locus, which leads to increased levels of the wild type α-synuclein, causes familial PD (Singleton et al 2003;Miller et al 2004).

Further the multiplication mutations, which are to date both triplication and duplication events appear to have a dose dependent effect on disease severity; thus triplications that double the genomic copy number of SNCA result in disease with an onset in the 4 th decade of life and duplications, which increase SNCA load by 50% result in disease onset in the 5 th or 6 th decade of life. Given this finding it is quite reasonable to suppose that common genetic variability at SNCA, which may increase expression by a small amount, is a risk factor for the

MAPT (encoding Microtubule associated protein tau)

There are six major brain isoforms of the microtubule associated protein tau From a pathological standpoint the relationship between tau and PD remains enigmatic: in general the brains of PD patients do not show abundant tau positive neuropathology; however the data supporting genetic association between MAPT and risk for PD continues to grow and is certainly one of the more robust findings in the field of risk variant neurogenetics.

GBA; Glucocerebrosidase

Glucocerebrosidase is a lysosomal enzyme that hydrolyses the beta-glycosidic linkage of glucosylceramide, a ubiquitous sphingolipid present in the plasma membrane of mammalian cells, originating ceramide and glucose. The human GBA gene is located on chromosome 1q21 and comprises 11 exons and 10 introns spanning over 7kb.

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A 5.5kb pseudogene, which shares over 96% homology with GBA, is located just 16kb downstream of the functional gene. The difference in size between the two is due o several Alu insertions in intronic regions of GBA. On the other hand, the lack of functionality of the pseudogene is attributed to two exonic deletions: a 4bp deletion in exon 4 and a 55bp deletion in exon 9. The pseudogene is absent in non-primate species and it has been suggested that the duplication event that originated the two genes, occurred about 40 million years ago. Interestingly, it has been shown that the orangutan doesn't present a pseudogene, but instead two functional genes, hence potentially four copies of GBA. and no mutations were found in the control samples. The percentage found in PD patients was of particular relevance, given that the carrier frequency for Gaucher disease-causing alleles is estimated at 0.006.

Mutations in

In In a smaller series of cases and controls collected in Venezuela (33 PD samples, 31 controls), Eblan M. et al. screened the entire coding region of the GBA gene and described an increase in mutation frequency among the early-onset PD samples when compared to the controls (12% vs 3.2%).

Toft M. et al published a report on the screening of two variants in GBA in individuals from Norway. This was the first report on northern European subjects.

The authors screened 311 PD patients and 474 healthy controls for the two common mutations N370S and L444P. They did not find an increased frequency of mutations in PD samples when compared to controls, however, the frequency of the mutations was surprisingly high when compared to estimates in white individuals (1.7% vs 0.6%). Though no statistical significance was obtained, the fact that only two variants were screened in a population not previously studied, may account for the lack of association.

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Several other studies reported positive associations of GBA mutations with PD, and one with Lewy bodies disorders. The most recent studies, which performed complete screenings of the gene, all found an association with PD in their study populations. Clark, L. et al. performed a study with two subsets of PD patients:

one with Jewish ancestry and another without Jewish background. Controls were also selected to match each of these groups. The frequency of GBA mutations is always greater in PD samples when compared to controls, particularly if only early-onset PD samples are considered.

Our group recently published a report on a cohort of Portuguese samples, where the enrichment of mutations among PD samples is also clear. This is the first report to obtain a clear statistically significant association in a population other than Ashkenazi Jewish. However, this same population has been shown to present an increased frequency of the G2019S mutation in the gene LRRK2; which a Jewish mutation.

An interesting result regarding GBA, and lysosomal enzymes in general, comes from the report of Balducci C. et al. who tested PD patients and controls for activity of lysosomal hydrolases in the CSF. GBA activity was significantly reduced in the CSF of PD patients, as would be expected by a loss-of-function model of mutations in these samples.

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Given all these results it seems clear that mutations in GBA are a risk factor for the development of PD, particularly early-onset PD. The mechanism by which mutations exert their effect and act as a risk factor is not yet understood, but several tentative explanations have been provided in the literature, relating to decrease in lysosomal function or involvement of the ubiquitin proteasome system.

Lrrk2

Mutations in LRRK2 were identified as a cause of PD in 2004 by us and others

[ref paisan-ruiz and zimprich]. A single mutation, G2019S, is a relatively common cause of PD in Caucasian populations, underlying approximately 2% of sporadic PD cases in North America and Northern Europe and 5% of cases with a positive family history for disease [lancet papers]. This mutation is more common in populations such as those from Portugal, those of Ashkenazi Jewish origin and from North African Arab populations; with this mutation underlying 8%, 21% and 41% of disease in these populations respectively. The G2019S does not however occur at appreciable frequency in control cohorts from these populations, so for the purpose of this article we will not designate this as a susceptibility variant.

Two variants reported from Asian populations however, do appear to be true risk variants for PD. The first G2385R was initially described in a Taiwanese family is present in PD populations at a frequency of ~10%, whereas it is only found in 0.5%-5% of controls. Taking a fairly conservative view of these results would suggest that carrying the risk allele imparts a two-fold risk increase in the chance of Parkinson's disease. Given that this association appears robust across Asian populations, this risk allele is an underlying factor in a very large number of PD cases worldwide.

More recently a second LRRK2 risk allele, also identified within Asian PD populations has been described.

OMI/HTRA2

Variants in the gene OMI/HTRA2 (OMIM #606441) were recently associated with an increased risk for PD [1]. This gene encodes a serine-protease with proapoptotic activity containing a mitochondrial targeting sequence at its Nterminal region. Several lines of evidence in the literature support a role for OMI/HTRA2 in neurodegeneration, the first of which was produced by Gray and colleagues [2] when they showed that Omi/HtrA2 interacts with presenilin-1, which is encoded by a gene known to be involved in Alzheimer's disease. The mutation initially thought to be pathogenic was found at the same frequency in PD samples and controls (0.77% and 0.72% respectively), indicating that it is not disease causing, but probably a rare variant in the German population.

Similarly for the p.A141S variant, no association with PD was found.

Evidence for the involvement of Omi/HtrA2 in neurodegenerative diseases are quite compelling at this point, but the genetic basis for this involvement is still not clear.

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examining this issue, has been hindered by the lack of studies that have performed full sequence and gene dosage analysis of PARK2 in large groups of cases and controls. Only a small number have studies have taken this approach and so far that data does not strongly support a large role for these mutations in typical PD, identifying pathogenic mutations in the heterozygous state in both cases and controls. This issue warrants a large scale sequencing effort, however, it is likely that

Genome Wide Association Studies

Genome wide association studies (GWAS) were a much-anticipated technology, and the application of this approach is expected to make major inroads into our understanding of the genetic basis of disease (REF SCIENCE). The basic tenet underlying GWAS is the common disease common variant hypothesis. A growing number of GWAS are being published and these are proving a valuable approach in understanding the genetics of complex disease. Two studies have been published thus far in PD; the success of these studies was limited somewhat by the sample size used, a point that is illustrated by the failure of these studies to identify SNCA or MAPT as potential risk loci. However, given the increasing investment in this technology (Manolio review) it is probable that several laboratories around the world are investing in large-scale GWAS in PD and that the next 2 years will see the identification of novel risk loci for this disease.

A

  form of the disease. In addition to the work characterizing the role of the REP alleles in risk for PD, more complete analyses have shown an association between genetic variability at other parts of SNCA with risk for disease; in particular variability in the 3' half of the gene (Mueller et al 2005; Mizuta et al 2006; Myhre et al 2008). Although this work does not resolve the issue of which are the pathobiologically relevant variants, they do suggests that investigation of REP alleles alone may miss, or underestimate, the contribution of genetic variability at SNCA to sporadic PD.

(

  hereafter called tau) generated by alternate splicing of exons 2, 3 and 10 of the gene MAPT. Alternate splicing of exon 10 results in tau with 3 or 4 microtubule binding respeats (3 repeat tau or 4 repeat tau). Tau is a major protein component of neurofibrillary tangles, a hallmark lesion of Alzheimer's disease; in these lesions tau appears to be deposited as a hyperphosphorylated insoluble filaments. Tau deposition is a hallmark of several other neurodegenerative disorders, including Picks disease (OMIM#), argyrophilic grain disease (OMIM#), corticobasal degeneration (OMIM#), progressive supranuclear palsy (PSP; OMIM#) and frontotemporal dementia (OMIM#). An initial link between the MAPT locus and disease was first reported in 1997 by Conrad and colleagues; who showed an association between a repeat A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT polymorphism close to the gene and PSP, indicating that variability at this locus is a risk factor for this disorder. In 1998 definitive evidence linking MAPT to neurodegenerative disease was provided when Hutton and colleagues identified mutations in this gene as a cause of frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP17). To date more than 35 mutations have been identified at the MAPT locus associated with this disease (for a good review see Haugorvoll, Wszolek, Hutton 2007). Many of these mutations are predicted to alter the alternative splicing of MAPT, altering the ratio of 3 repeat tau to 4 repeat tau (Hutton, Lendon et al; and Spillantini, Murrell et al). In addition to rare causal mutations, common variability in MAPT has been linked to disease; notably robust association between MAPT and risk for PSP, AD and most recently PD has been reported. From a genomic perspective the architecture of the MAPT locus is unusual; the gene sits within a large block approximately 1.6 million base pairs long that shows reduced recombination and thus high levels of linkage disequilibrium. This appears to be a result of a common genomic inversion in the Caucasian population; this inversion inhibits recombination between genomic fragments that are in the opposite orientation. This phenomenon results in two common Caucasian haplotype groups across this locus; often termed H1 and H2. Association between MAPT H1 subhaplotypes and risk for PD has been tested by many groups (REFS FROM PD GENE), and the results in general show a consistent association with disease, the H1 haplotype conferring a risk with an odds ratio of approximately 1.3 (for A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT summary statistics see the PD Gene database http://www.pdgene.org/meta.asp?geneID=14). Evidence is also mounting that the MAPT risk alleles for these disorders are associated with increased MAPT expression; either in total or specific to four-repeat tau splice variants (i.e. those containing exon 10) (Caffrey et al HMG; Myers and Pittman). Most recently Tobin and colleagues (REF) have shown association between PD risk and a subhaplotype of H1; these authors then extended this work to show over-expression of 4 repeat tau in the brains of PD patients.

  GBA are the cause of a recessive lysosomal storage disorder -Gaucher disease. Patients with Gaucher present macrophages enlarged with deposits of glucosylceramide, suggesting that mutations in GBA act in a loss-offunction fashion. Over 200 mutations have been described in GBA, including point mutations, deletions and recombination alleles derived from the pseudogene sequence. It has been estimated that approximately 20% of the pathogenic mutations in GBA are caused by recombination or gene conversion between the two genes. Although mutations are distributed over the entire GBA coding region, pathogenic mutations seem to cluster in the carboxyl-terminal region, which encodes the catalytic domain. Phenotypes of Gaucher and Parkinson's diseases do not overlap significantly, but the first indication for a relationship between the two, actually came from A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT clinical descriptions. These reported patients with Gaucher disease who developed early-onset, treatment-refractory parkinsonism. The first report of an increased frequency of mutations in GBA in Parkinson disease patients was published in 2003. Here, the authors screened 57 brain samples from subjects with PD and 44 brain samples from adult subjects without a diagnosis of PD. Mutations in GBA were identified in 14% of the PD samples,

(

  Mata et al 2005; neurogenetics). Assessment of this variant in large Asian populations showed association with risk for disease in Taiwanese (DiFonzo et al 2005; Fung et al 2006; Farrer et al 2007), Japanese (Funayama et al 2007), (Chan et al 2008) and mainland Chinese (An et al 2008; Li et al 2007) populations. In general this work showed that the risk variant, 2385R

  Moreover beta-amyloid, which plays a pivotal role in the pathogenesis of Alzheimer's, was shown to be cleaved by Omi/HtrA2 [3]. Mouse models also provided support for the involvement of this protein in neurodegeneration; a A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT mutation in the protease domain of Omi/HtrA2 was found as the genetic cause underlying the disease in the mnd2 mutant mouse [4] and the knockout mouse showed loss of neurons in the striatum concomitant with parkinsonian features [5]. The description of mutations associated with PD came from the work of Strauss and colleagues in 2005 [1]. Here, they screened a large cohort of 518 German PD patients and 370 healthy control individuals for mutations in OMI/HTRA2.One variant (p.G399S) was found only in PD patients (n=4) suggesting that it would be a pathogenic mutation. The other variant (p.A141S) was found significantly overrepresented in the PD group (p=0.039) suggesting that it would act as a risk factor for PD. In vitro studies of both variants provided evidences of functional roles. Additionally, Omi/HtrA2 was detected in Lewy bodies in brain tissue from PD patients.More recently, Simon-Sanchez and Singleton [6] presented a thorough analysis of the coding region of OMI/HTRA2 in a case-control study, which comprised a large cohort of PD patients (n=644) and neurologically normal controls (n=828).

  gene PARK2, was the second genetic cause of parkinsonism identified. Mutation of this gene was found to cause an autosomal recessive juvenile form of parkinsonism. As with many autosomal recessive diseases, most pathogenic mutations are loss of function variants, involving large structural genomic disruption of the coding region of the gene or premature termination of the transcript; in addition several missense mutations have been identified with varying levels of proof vis a vis association with disease. More controversial still, is the role of single heterozygous mutations as risk factors for late onset typical PD. Much of this work has been driven by observations in family based studies where later onset typical PD was seen in relatives of patients with young onset parkinsonism -analysis of these cases revealed homozygous or compound heterozygous PARK2 mutations in the young-onset cases and possession of single PARK2 mutations in the later onset affected relatives; such studies have been criticized because of the significant confound of ascertainment bias; i.e. the families that tend to be collected and analyzed are those with many affected family members. Further support for the role of single PARK2 mutations as a risk factor for disease is the observation that heterozygous mutation carriers display dopamine reuptake deficiency in fdopa PET analysis (KHAN ET AL). The strength of the case for a role of heterozygous PARK2 mutations as risk loci based on these observations, and on data arising from case control analysesA C C E P T E DM A N U S C R I P T

  large sample series they necessitate inter-laboratory collaborations and large consortia, individual investigators are by and large, unable to accumulate large enough series. Clearly the formation of such collaborations is a good thing for research; they facilitate communication between scientists, maximize the chances of finding positive signals and engender trust between research groups that allows collaboration outside the immediate aims of the collaborative framework. This latter point is particularly critical; as technologies improve GWAS will be expanded to include better genomic coverage (currently even the most dense SNP platforms probably only capture ~70% of common variation) in more samples TABLE WITH VARIANTS< OR< POPULATION STUDIED< FREQUENCYA C C E P T E D M A N U S C R I P T

  only included sample sizes in the low hundreds. Early genome wide association data shows quite convincingly that in terms of common genetic risk factors, there are none with an odds ratio greater than two in the North American White population. It is likely that sample sizes of more than a thousand are required to detect effects of this size. The second limitation is a Thus the majority of previous studies were not only unlikely to have selected a

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	question, however these two findings elegantly linked these disease entities; this
	work simply that the mutant form of α-synuclein causes a familial form of PD and
	M A N U S C R I P T that the α-synuclein protein is a component of the pathology of all cases of PD. Shortly after this work Krüger and colleagues reported an association between common genetic variability in SNCA and risk for PD, specifically an association with an imperfect dinucleotide repeat approximately 10kb 5' to the translation start site of SNCA. Variability at and proximal to REP has been examined in a large number of studies (Farrer et al 2001; Tan et al 2003; Pals et al 2004; Mellick et al 2005; Hadjigeorgiou et al 2006; Khan et al 2001; Spadafora et al M A N U S C R I P T 2003; Izumi et al 2001) and most recently a meta-analysis of published studies
	A C C E P T E D and combination with novel data revealed a consistent association between risk for disease and the longer REP allele (Maraganore et al, XXX). Experimental in A C C E P T E D vitro evidence suggests that this allele is associated with increased expression of
	cause of a rare familial form of PD. Closely after the initial identification of the first
	disease cause mutation in SNCA the protein product of this gene, α-synuclein,
	was shown to be a major constitutive part of the pathologic hallmark inclusion of
	PD, the Lewy body. The relevance of studying rare familial forms of PD to
	understanding the common non-familial form of this disease had remained in

generally genuine risk locus or variant for interrogation but further were likely not powered to detect risk effects, should they exist. The exception to this work is that which has centered on exhaustive analysis of genes already implicated in familial forms of related disorders; such analyses provide two of the most convincing sets of genetic association data for PD, implicating the genes SNCA and MAPT as risk loci.

SNCA (encoding α-synuclein)

Genetic variability at SNCA is arguably the most reliable association of a common genetic risk locus with PD identified to date. The impetus for examination of this locus resulted from the cloning of SNCA mutations as a


Juan Carlos Molero

Scott Lee

Ilit Leizerman

Ayelet Chajut

Adrian Cooper

Ken Walder
email: walder@deakin.edu.au

Effects of rosiglitazone on intramyocellular lipid accumulation in Psammomys obesus

Keywords: intramyocellular lipid, rosiglitazone, thiazolidinedione, Psammomys obesus

Objective: To examine the effects of rosiglitazone in intramyocellular lipid (IMCL) content in diabetic P. obesus using novel electron microscopy technologies. Background: P. obesus is an unique polygenic model of obesity and type 2 diabetes. Male diabetic P. obesus were treated daily with 5 mg/Kg Rosiglitazone by oral gavage for 14 days. Data were compared with a group of age-matched diabetic P. obesus treated with saline vehicle.

Methods: Assesment of insulin resistance and adiposity were determine before and after the treatment period by oral glucose tolerance test (oGTT) and dual energy Xray absorptiometry (DEXA) analysis. We used a new scanning electron microscopy technology, (WETSEM) to investigate the effects of rosiglitazone administration on IMCL content, size and distribution in red gastrocnemious muscle.

Results: Rosiglitazone treatment improved glucose tolerance in P. obesus with no difference in the overall body fat content although a significant reduction in subscapular fat mass was observed. Rosiglitazone changed the distribution of lipid droplet size in skeletal muscle. Treated animals tended to have smaller lipid droplets compared with saline-treated controls.

Conclusions: Since smaller IMCL droplets are associated with improvements in insulin sensitivity, we propose that this may be an important mechanism by which rosiglitazone affects glucose tolerance.

A C C E P T E D M A N U S C R I P T Introduction

Thiazolidinediones (TZD's) such as rosiglitazone improve whole body insulin sensitivity in both rodents [START_REF] Oakes | A new antidiabetic agent, BRL 49653, reduces lipid availability and improves insulin action and glucoregulation in the rat[END_REF][START_REF] Young | Repeat treatment of obese mice with BRL 49653, a new potent insulin sensitizer, enhances insulin action in white adipocytes. Association with increased insulin binding and cellsurface GLUT4 as measured by photoaffinity labeling[END_REF] and humans with insulin resistance [START_REF] Nolan | Rosiglitazone taken once daily provides effective glycaemic control in patients with Type 2 diabetes mellitus[END_REF][START_REF] Phillips | Once-and twice-daily dosing with rosiglitazone improves glycemic control in patients with type 2 diabetes[END_REF]. TZD's act in a range of tissues to improve insulin sensitivity including adipose tissue [START_REF] Ibrahimi | Evidence for a common mechanism of action for fatty acids and thiazolidinedione antidiabetic agents on gene expression in preadipose cells[END_REF], skeletal muscle [START_REF] Oakes | A new antidiabetic agent, BRL 49653, reduces lipid availability and improves insulin action and glucoregulation in the rat[END_REF][START_REF] Oakes | The insulin sensitizer, BRL 49653, reduces systemic fatty acid supply and utilization and tissue lipid availability in the rat[END_REF][START_REF] Hevener | Muscle-specific Pparg deletion causes insulin resistance[END_REF], liver [START_REF] Oakes | A new antidiabetic agent, BRL 49653, reduces lipid availability and improves insulin action and glucoregulation in the rat[END_REF][START_REF] Oakes | The insulin sensitizer, BRL 49653, reduces systemic fatty acid supply and utilization and tissue lipid availability in the rat[END_REF] and macrophages [START_REF] Hevener | Macrophage PPAR gamma is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones[END_REF]. TZD's are also thought to have protective effects in pancreatic β-cells [START_REF] Sanchez | Effect of rosiglitazone on the differential expression of diabetes-associated proteins in pancreatic islets of C57Bl/6 lep/lep mice[END_REF]. The principle mechanism of action of TZD's is thought to be through agonism of peroxisome proliferator-activated receptor-gamma (PPARγ), leading to transcriptional regulation of a number of genes in various tissues. Collectively, these transcriptional changes are thought to cause a re-distribution of lipids from skeletal muscle and liver to adipose tissue. Together with increased adipogenesis, this results in increased storage of lipids in smaller, more insulin sensitive adipocytes, leading to an overall improvement in insulin sensitivity [START_REF] Lehmann | An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma)[END_REF][START_REF] Shulman | Cellular mechanisms of insulin resistance[END_REF].

Recent studies have demonstrated the importance of inflammation in the development of insulin resistance [START_REF] Yuan | Reversal of obesity-and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta[END_REF][START_REF] Hundal | Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes[END_REF][START_REF] Arkan | IKK-beta links inflammation to obesity-induced insulin resistance[END_REF]. TZD's have been shown to have significant antiinflammatory effects in a range of in vitro and in vivo models, and in human subjects [START_REF] Ricote | The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation[END_REF][START_REF] Jiang | PPAR-gamma agonists inhibit production of monocyte inflammatory cytokines[END_REF][START_REF] Mohanty | Evidence for a potent antiinflammatory effect of rosiglitazone[END_REF][START_REF] Van Doorn | Evaluation of proinflammatory cytokines and inflammation markers as biomarkers for the action of thiazolidinediones in Type 2 diabetes mellitus patients and healthy volunteers[END_REF], leading to some conjecture regarding the principal mechanism of action by which TZD's improve insulin sensitivity.

Accumulation of triglycerides in skeletal muscle is strongly associated with insulin resistance, and IMCL content is associated with both insulin resistance and type 2 diabetes [START_REF] Oakes | The insulin sensitizer, BRL 49653, reduces systemic fatty acid supply and utilization and tissue lipid availability in the rat[END_REF][START_REF] Storlien | Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid[END_REF][START_REF] Kraegen | Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats[END_REF][START_REF] Krssak | Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study[END_REF][START_REF] Pan | Skeletal muscle triglyceride levels are inversely related to insulin action[END_REF][START_REF] Kelley | Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss[END_REF]. However a paradox has been described whereby highly trained endurance athletes have high levels of IMCL but are very healthy and highly insulin sensitive [START_REF] Russell | Lipid peroxidation in skeletal muscle of obese as compared to endurance-trained humans: a case of good vs. bad lipids?[END_REF][START_REF] Goodpaster | Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes[END_REF][START_REF] Bruce | Disassociation of muscle triglyceride content and insulin sensitivity after exercise training in patients with Type 2 diabetes[END_REF]. It appears that it is not only the amount of lipid in skeletal muscle but the size and distribution of the lipid droplets that determines the level of insulin sensitivity in this organ.

While it is clear that the accumulation of IMCL is strongly associated with insulin resistance and the risk of type 2 diabetes, the effects of rosiglitazone on IMCL levels have been inconsistent. Several studies have shown that rosiglitazone reduced IMCL in conjunction with improved insulin sensitivity [START_REF] Kim | Differential effects of rosiglitazone on skeletal muscle and liver insulin resistance in A-ZIP/F-1 fatless mice[END_REF][START_REF] Jucker | Reduction of intramyocellular lipid following short-term rosiglitazone treatment in Zucker fatty rats: an in vivo nuclear magnetic resonance study[END_REF][START_REF] Koh | Peroxisome proliferator-activated receptor (PPAR)-alpha activation prevents diabetes in OLETF rats: comparison with PPAR-gamma activation[END_REF][START_REF] Hockings | Rapid reversal of hepatic steatosis, and reduction of muscle triglyceride, by rosiglitazone: MRI/ S studies in Zucker fatty rats[END_REF][START_REF] Kuhlmann | Intramyocellular lipid and insulin resistance: a longitudinal in vivo 1H-spectroscopic study in Zucker diabetic fatty rats[END_REF], however a number of other studies have found no change [START_REF] Mayerson | The effects of rosiglitazone on insulin sensitivity, lipolysis, and hepatic and skeletal muscle triglyceride content in patients with type 2 diabetes[END_REF] or an increase in IMCL following treatment with rosiglitazone [START_REF] Lessard | Rosiglitazone enhances glucose tolerance by mechanisms other than reduction of fatty acid accumulation within skeletal muscle[END_REF][START_REF] Muurling | Rosiglitazone improves muscle insulin sensitivity, irrespective of increased triglyceride content, in ob/ob mice[END_REF].

The aim of the current study was to examine the effects of rosiglitazone treatment in Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes. In their natural semi-arid habitat, these gerbil-like rodents feed on low energy plants such as saltbush (Atriplex halimus). However, when fed a high caloric diet in captivity, they develop aspects of the metabolic syndrome characterised by obesity, insulin resistance and hyperglycaemia. [START_REF] Barnett | A cross-sectional and short-term longitudinal characterisation of NIDDM in Psammomys obesus[END_REF][START_REF] Walder | Characterization of obesity phenotypes in Psammomys obesus (Israeli sand rats)[END_REF]. To address the aim of this study we have utilised a novel electron microscopy technology to investigate the effects of rosiglitazone on IMCL content, size and distribution in skeletal muscle.

Materials and Methods

Animals

Study protocol

Two experimental groups (saline or rosiglitazone treated) of 16-20 week-old animals were matched for body weight, food consumption and circulating glucose and insulin levels (see table 1). Both groups of animals were hyperinsulinemic but normoglycaemic at the beginning of the study. Each group were treated by daily oral gavage of rosiglitazone (5 mg/kg) or saline (control) for 14 d. Body weight and food intake were measured weekly. Blood samples were collected for assessment of circulating glucose and insulin levels on days 0, 7 and 14. At the start and end of the study, all animals had energy homoeostasis assessed by indirect calorimetry (24 h), and body composition measured by DEXA analysis. An oral glucose tolerance test (2 g/kg glucose) was performed on day 14.

WETSEM technology: background

Red gastrocnemius muscle was harvested and immersed in formalin, and longitudinally cut to a thickness of 0.5-1 mm, and stained with phosphtungstic acid (PTA; Sigma, St. Louis, MO), which highlights proteins and membranous structures. Using this staining methodology, intracellular features were highlighted brightly whereas lipid droplets remained unstained in black.

The WETSEM technology is especially useful to resolve lipid compartments in the cell, based on their rich carbon content compared with other cellular structures richer in oxygen atoms. The WETSEM technique was described recently in detail [START_REF] Thiberge | Scanning electron microscopy of cells and tissues under fully hydrated conditions[END_REF][START_REF] Nyska | Electron microscopy of wet tissues: a case study in renal pathology[END_REF][START_REF] Nyska | A new method of wet scanning electron microscopy for the analysis of myelination in EAE mouse model of multiple sclerosis[END_REF]. Briefly, each stained muscle section was absorbed onto a thin partition membrane transparent to electrons inside a sample capsule (QuantomiX Ltd. Nes Ziona, Israel) and placed on the specimen stage of the SEM under vacuum. Images were obtained on an FEI XL-30 (Eindhoven, The Netherlands) scanning electron microscope (electron beam energy of 15-30 KV, beam current of 200-800 pA, with scan rates of 1.3-120 msec/line at 484 lines/frame).

Assessment of lipid accumulation in muscle

For each animal, a total of 40 fibers were imaged, representing different longitudinal muscle fibers per sample. Determination of IMCL droplet size and total amount was performed using custom-made in-house software (QuantomiX, Nes Ziona, Israel). Typical resolution of choice for image processing was as shown in Figure 3.

Statistical analysis

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Results

Effects of rosiglitazone in Psammomys obesus

Treatment with rosiglitazone had no effect on body weight, percentage body fat (assessed by DEXA) or plasma insulin concentration in P. obesus (Table 1). However, rosiglitazone treatment caused a significant reduction in mean blood glucose concentration (p=0.006), and was associated with improved glucose tolerance as measured by OGTT (Figure 1). During the OGTT, blood glucose concentrations were significantly lower in rosiglitazone treated P. obesus compared with controls after 45 min (p= 0.04), 60 min (p=0.04) and 90 min (p=0.01), consistent with an improvement in glucose tolerance following treatment with rosiglitazone. Furthermore, after 14 days of rosiglitazone treatment (5 mg/kg/d), the area under the glucose curve in an OGTT was reduced by 19% (p=0.039) compared with controls (Figure 1B).

There was no difference in the weights of major organs (liver, heart, pancreas, spleen, heart, adrenal gland, and testis) in the rosiglitazone treated animals compared with saline treated controls. Consistent with the results of the DEXA analysis of body fat content, the combined weight of the various adipose tissue depots was not different between the groups (rosiglitazone 12.0±1.2 vs. saline 14.7±1.7 g, p=0.20). In the animals treated with rosiglitazone, the weight of the sub-scapular (subcutaneous) fat pad was reduced by 38% when compared with the saline treated control animals (Figure 2, p=0.025). This difference remained when the sub-scapular fat pad mass was expressed as a percentage of body weight (rosiglitazone 1.24±0.11 v saline 1.92±0.27 % of body weight, p=0.020).

There was no significant difference in the mass (or percentage of body weight) for any of the other adipose depots tested (peri-renal, epididymal, mesenteric or intramuscular; Figure 2), nor for any of the skeletal muscles measured (data not shown).

Treatment of P. obesus with rosiglitazone had no effect on total energy expenditure, physical activity, or whole-body fat or carbohydrate oxidation as measured by indirect calorimetry in P. obesus.

Imag ing and quantitative analysis of IMCL

Figure 3 shows a series of representative muscle images taken at different magnifications, using the WETSEM technique. The high-resolution images of the muscle fibers, showing the precise structure of the striated skeletal muscle, the IMCL droplets and the mitochondria, enabled determination of IMCL droplet size.

Total IMCL content per fibre area was not significantly different between the treatment groups, although IMCL tended to be lower in the rosiglitazone treated animals (Fig. 4A). Analysis of IMCL droplet size distribution indicated a pattern of reduced lipid droplet size in the skeletal muscle of P. obesus treated with rosiglitazone

A B D C

(shown in black bars) compared with control animals (white bars; Fig. 4B). There was significantly reduced incidence of larger lipid droplets in the rosiglitazone treated animals, relative to the controls (χ 2 =9.9, p<0.05).

Correlation between physiological parameters and IMCL analysis

We used multiple regression analysis to investigate relationships between physiological data and the IMCL data set obtained from the WETSEM technique. When all animals were analysed together, a combination of the area under the curve of the OGTT results (OGTT-AUC) and the percentage body fat at day 14 was significantly associated with IMCL droplet size (R 2 = 0.44). Within the saline treated animals, OGTT-AUC (p=0.02) and fat content at day 14 (p=0.009) were strongly and independently associated with IMCL droplet size (R 2 = 0.74).

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Discussion

In this study we have shown that treatment with rosiglitazone (5 mg/kg) for 14 days significantly improved glucose tolerance in P. obesus. The improvement in glucose tolerance was not associated with a change in total IMCL, but there was a trend for a reduction in lipid droplet size in skeletal muscle following rosiglitazone treatment. Furthermore, we demonstrated that IMCL was significantly associated with glucose tolerance and overall body fat content. Collectively, these data suggest that the effect of rosiglitazone on glucose tolerance may be mediated in part by changing the distribution of lipid droplet size in skeletal muscle, even if there is no overall reduction in total IMCL.

Treatment with rosiglitazone did not affect body weight or body fat content in P. obesus. However, the mass of the sub-scapular fat pad was reduced in the treated animals compared with controls. There was no difference between the groups for any of the other fat pads measured, and the relevance of this finding to the overall effects of rosiglitazone on glucose tolerance is unknown. However, this finding does provide additional evidence to the suggestion that treatment with rosiglitazone results in redistribution of lipids between tissues.

The relationship between IMCL and insulin resistance is well established [START_REF] Storlien | Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid[END_REF][START_REF] Kraegen | Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats[END_REF][START_REF] Krssak | Intramyocellular lipid concentrations are correlated with insulin sensitivity in humans: a 1H NMR spectroscopy study[END_REF][START_REF] Pan | Skeletal muscle triglyceride levels are inversely related to insulin action[END_REF][START_REF] Kelley | Skeletal muscle fatty acid metabolism in association with insulin resistance, obesity, and weight loss[END_REF].

However, the effects of rosiglitazone on IMCL are less clear [START_REF] Mayerson | The effects of rosiglitazone on insulin sensitivity, lipolysis, and hepatic and skeletal muscle triglyceride content in patients with type 2 diabetes[END_REF][START_REF] Lessard | Rosiglitazone enhances glucose tolerance by mechanisms other than reduction of fatty acid accumulation within skeletal muscle[END_REF][START_REF] Muurling | Rosiglitazone improves muscle insulin sensitivity, irrespective of increased triglyceride content, in ob/ob mice[END_REF], and there is some conjecture regarding the mechanisms by which this drug improves insulin sensitivity. Here we provide evidence to suggest that rosiglitazone treatment results in a reduction in lipid droplet size in skeletal muscle without a significant change in total IMCL. Hefetz and colleagues also reported no change in the total lipid content in skeletal muscle from diabetic P. obesus following rosiglitazone treatment, although they did not examine the size distribution of the lipid droplets [START_REF] Hefetz | Prevention of nutritionally induced diabetes by rosiglitazone in the gerbil Psammomys obesus[END_REF]. Interestingly, microscopy analysis of pancreatic sections revealed that rosiglitazone prevented the decrease in insulin immunostaining induced by the high energy diet and accelerated β-cell proliferation (measured by BrdU incorporation into nuclei) [START_REF] Hefetz | Prevention of nutritionally induced diabetes by rosiglitazone in the gerbil Psammomys obesus[END_REF]. Based on these observations, Hefetz et al. concluded that the major anti-diabetic action of rosiglitazone in P. obesus was by preventing the deterioration of pancreatic function. While we have not formally examined insulin content in β-cell islets in this study, we observed no change in circulating insulin levels after rosiglitazone treatment. This suggests that the beneficial effects of rosiglitazone on glycaemia and glucose tolerance shown in this study are not likely due to an increase in insulin secretion. The alternative conclusions reached in both studies may be due to differences in the dose of rosiglitazone and the high calorie feeding protocol used.

We believe that a reduction in lipid droplet size in skeletal muscle may be an important factor in the improvement in glucose tolerance observed in these animals Consistent with this hypothesis, Fraenkel et al showed that a decrease in the number of large lipid droplets in skeletal muscle was associated with reduced hyperglycaemia when high energy diet induced-diabetic P. obesus were switched to a low energy diet [START_REF] Fraenkel | Scanning electron microscopic analysis of intramyocellular lipid droplets in an animal model of type 2 diabetes[END_REF]. Likewise, He and colleagues [START_REF] He | Effects of weight loss and physical activity on muscle lipid content and droplet size[END_REF] showed a profound increase in insulin sensitivity following exercise training and weight loss in obese subjects, with no change in total IMCL. However the size of intramuscular lipid droplets was significantly reduced and this reduction was correlated with insulin sensitivity [START_REF] He | Effects of weight loss and physical activity on muscle lipid content and droplet size[END_REF]. One potential mechanism by which a reduction in lipid droplet size may be associated

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with insulin sensitivity independent of total IMCL is through an increased surface to volume ratio of the droplets, which increases the proportion of PAT proteins (perilipin, adipophilin, and TIP47) relative to triglyceride content, in turn improving the access of cytosolic lipases to the lipids [START_REF] Hesselink | Intramyocellular lipids and insulin sensitivity: does size really matter?[END_REF]. It has also been suggested that smaller lipid droplets are more accessible to oxidation by mitochondria [START_REF] He | Effects of weight loss and physical activity on muscle lipid content and droplet size[END_REF].

The improvement in glucose tolerance with reductions in lipid droplet size but no change in total IMCL is also consistent with the "athletes paradox". Highly trained endurance athletes have high levels of IMCL but are highly insulin sensitive [START_REF] Russell | Lipid peroxidation in skeletal muscle of obese as compared to endurance-trained humans: a case of good vs. bad lipids?[END_REF][START_REF] Goodpaster | Skeletal muscle lipid content and insulin resistance: evidence for a paradox in endurance-trained athletes[END_REF][START_REF] Bruce | Disassociation of muscle triglyceride content and insulin sensitivity after exercise training in patients with Type 2 diabetes[END_REF].

These observations have been explained by the fact that oxidative capacity is greatly increased following exercise training, although the precise role of lipid droplet size and subcellular location have not been fully explored. Our results, along with those of others [START_REF] He | Effects of weight loss and physical activity on muscle lipid content and droplet size[END_REF] suggest that lipid droplet size may be an important determinant of insulin sensitivity of skeletal muscle, and that this is one mechanism by which rosiglitazone can affect glucose tolerance.

In summary, rosiglitazone improved glucose tolerance in P. obesus, and this effect was associated with a re-distribution of lipid droplet size in skeletal muscle. We hypothesise that this may an important aspect of the mechanism of action of rosiglitazone, and could explain some of the inconsistent results seen previously regarding the effects of rosiglitazone on IMCL. Future studies should include assessment of not only total IMCL, but also the size of the lipid droplets in skeletal muscle.

Figure 1 :

 1 Figure LegendsFigure 1: A) OGTT blood glucose data showing that rosiglitazone improves glucose tolerance in P. obesus. *p<0.05 compared with saline treated control animals. B) Area under the blood glucose curve (OGTT) data showing an improvement in glucose tolerance following rosiglitazone treatment in P. obesus. *p=0.039.Figure 2: Fat pad weights from P. obesus treated with rosiglitazone or saline (control). *p=0.025.Figure 3: Wet SEM images of skeletal muscle from P. obesus treated with rosiglitazone (C and D) or saline (controls; A and B). Scale bars are shown in the lower right corner of each image.Figure 4: A) Total IMCL per fibre area in P. obesus treated with rosiglitazone and saline. B) IMCL droplet size distribution (normalised to the mean per 10 fibres) in rosiglitazone (black bars) and saline (control; white bars) treated P. obesus. χ2=9.9, p<0.05.

Figure 2 :

 2 Figure LegendsFigure 1: A) OGTT blood glucose data showing that rosiglitazone improves glucose tolerance in P. obesus. *p<0.05 compared with saline treated control animals. B) Area under the blood glucose curve (OGTT) data showing an improvement in glucose tolerance following rosiglitazone treatment in P. obesus. *p=0.039.Figure 2: Fat pad weights from P. obesus treated with rosiglitazone or saline (control). *p=0.025.Figure 3: Wet SEM images of skeletal muscle from P. obesus treated with rosiglitazone (C and D) or saline (controls; A and B). Scale bars are shown in the lower right corner of each image.Figure 4: A) Total IMCL per fibre area in P. obesus treated with rosiglitazone and saline. B) IMCL droplet size distribution (normalised to the mean per 10 fibres) in rosiglitazone (black bars) and saline (control; white bars) treated P. obesus. χ2=9.9, p<0.05.

Figure 3 :

 3 Figure LegendsFigure 1: A) OGTT blood glucose data showing that rosiglitazone improves glucose tolerance in P. obesus. *p<0.05 compared with saline treated control animals. B) Area under the blood glucose curve (OGTT) data showing an improvement in glucose tolerance following rosiglitazone treatment in P. obesus. *p=0.039.Figure 2: Fat pad weights from P. obesus treated with rosiglitazone or saline (control). *p=0.025.Figure 3: Wet SEM images of skeletal muscle from P. obesus treated with rosiglitazone (C and D) or saline (controls; A and B). Scale bars are shown in the lower right corner of each image.Figure 4: A) Total IMCL per fibre area in P. obesus treated with rosiglitazone and saline. B) IMCL droplet size distribution (normalised to the mean per 10 fibres) in rosiglitazone (black bars) and saline (control; white bars) treated P. obesus. χ2=9.9, p<0.05.

Figure 4 :

 4 Figure LegendsFigure 1: A) OGTT blood glucose data showing that rosiglitazone improves glucose tolerance in P. obesus. *p<0.05 compared with saline treated control animals. B) Area under the blood glucose curve (OGTT) data showing an improvement in glucose tolerance following rosiglitazone treatment in P. obesus. *p=0.039.Figure 2: Fat pad weights from P. obesus treated with rosiglitazone or saline (control). *p=0.025.Figure 3: Wet SEM images of skeletal muscle from P. obesus treated with rosiglitazone (C and D) or saline (controls; A and B). Scale bars are shown in the lower right corner of each image.Figure 4: A) Total IMCL per fibre area in P. obesus treated with rosiglitazone and saline. B) IMCL droplet size distribution (normalised to the mean per 10 fibres) in rosiglitazone (black bars) and saline (control; white bars) treated P. obesus. χ2=9.9, p<0.05.

ACCEPTED MANUSCRIPTThe relationship between the dependent variable from the IMCL data, and several independent variables from the blood biochemical data was analyzed by multiple regression analysis using SAS software.

Acknowledgments

This work was supported by Verva Pharmaceuticals Ltd (Australia). We thank Dr. Daniel Rothenstein for assistance with the statistical analysis.

A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T


Manildo M Oliveira

Moacelio V Silva Filho

Vera L F Cunha Bastos

Flavio C Fernandes

Jayme Cunha Bastos
email: jbastos@uerj.br

Jayme Cunha Bastos

Brain acetylcholinesterase as a marine pesticide biomarker using Brazilian fishes

Keywords: acetylcholinesterase, organophosphate, pollutant, biomarker

Brain acetylcholinesterase (AChE) of some fishes from the coast of Rio de Janeiro state was studied as a possible pesticide biomarker in marine environmental monitoring. AChE specific activity in brain varied from 145 to 530 U/g of proteins and the Michaelis-Menten constant (K M ) for acetylthiocholine varied from 104 to 291 µM among the twenty species studied. The enzyme sensitivity to methyl paraoxon, evaluated by the inhibition kinetic constants, shows that some species (Paralonchurus brasiliensis and Genidens genidens) are more sensitive (IC50-30min = 455 and 468 nM, respectively). The less sensitive Merluccius hubbsi and Percophis brasiliensis (IC50-30min = 3,339 and 3,259 nM, respectively) belong to the super-order Paracanthopterygii, which includes the more ancient species. On the other hand, more susceptible species belong to the super-order Acanthopterygii, which includes more recent species. These results suggest a possible evolutionary linkage for AChE sensitivity to methyl paraoxon. The application of inhibition kinetic constants for fish brain AChE in phylogenetic studies is still being investigated. The results have shown that a fish sentinel species should have the highest brain AChE level among the more sensitive ones.

Introduction

Pesticides are used in agriculture to control insects and other pests, which contaminates rivers and, eventually, the sea. In addition to traditional chemical analysis, activity of the enzyme acetylcholinesterase (AChE) has been used for environmental monitoring programs (Burgeot, Bocquéné, Porte, Dimet, Santella, Garcia de la Parra et al., 1996;[START_REF] Huang | Evaluation of liver and brain esterases in the spotted gar fish (Lepisosteus oculatus) as biomarkers of effect in the lower Mississippi River basin[END_REF]; Minier, Levy, Rabel, Bocquéné, Godefroy, Burgeot et al., 2000) because AChE inhibition in tissues of fish and other animals can be used as biomarker for organophosphorous and carbamates exposure [START_REF] Escartin | Acetylcholinesterase inhibition in the crayfish procarambus clarkii exposed to fenitrothion[END_REF][START_REF] Sturn | Cholinesterases of marine teleost fish: enzymological characterization and potencial use in the monitoring of neurotoxic contamination[END_REF]. Inhibition of AChE is not necessarily fatal, but using a fish with a highly sensitive AChE as a sentinel species will allow the detection of lower contamination levels.

The proper use of enzymatic biomarkers requires a fundamental understanding of its biochemistry for the species to be utilized. Vertebrate cholinesterases have been proposed to exist alternatively as globular forms or asymmetric forms associated with a collagen-containing tail [START_REF] Massoulié | Molecular and cellular biology of cholinesterases[END_REF]. Soluble globular forms may be nonamphiphilic or amphiphilic [START_REF] Bon | Amphiphilic and nonamphiphilic forms of Torpedo cholinesterases: I. Solubility and Aggregation Properties[END_REF]. Because asymmetric forms of vertebrate cholinesterases require highsalt buffers for solubilization, whereas amphiphilic forms require detergent for solubilization, inaccurate measurements of ChE activity may result from the inability to consistently and thoroughly extract the enzyme from tissue samples.

In addition, it is important to consider how environmental history and seasonal conditions affect the physiology of the sentinel species [START_REF] Monserrat | Kinetic and toxicological characteristics of acetylcholinesterase from the gills of oysters (Crassostrea rhizophorae) and other aquatic species[END_REF]Rodrıguez-Fuentes & Gold-Bouchot, 2004).

The present work is part of a marine environmental monitoring program (MOMAM)

undertaken by the Instituto de Estudos do Mar Almirante Paulo Moreira (IEAPM) of the Brazilian Navy. The aim of this work was to assess the potential of brain AChE of some coastal marine fish in Rio de Janeiro State as a biomarker for organophosphates and carbamates.

Materials and Methods

The oceanographic studies were carried out along the coast of Rio de Janeiro State, in Southeastern Brazil, as shown in Table 1 andFigure 1. Seawater samples were collected with a Nansen bottle and assayed for dissolved oxygen, nitrate, ammonia and phosphate according to [START_REF] Strickland | A Practical Handbook of Seawater Analysis[END_REF]. The salinity was measured with a density-meter model RS-23C (Beckman). The temperatures were measured with thermometers on the Nansen bottle.

The ocean specimen captures were carried out using an otter-trawl in an IEAPM boat, dragging for 20 min at 3 knots. Fish from the Sepetiba Bay were caught by fishermen with rod, line and hook. The animals were identified according to [START_REF] Figueiredo | Manual de Peixes Marinhos do Sudeste do Brasil[END_REF] and were sacrificed in situ. Their brains were removed, rapidly bathed in a cold 0.9% NaCl solution and wrapped in parafilm. Each small bag was thrown in liquid nitrogen and then kept frozen at -20 ºC in a freezer until homogenization. After being defrosted, brains were mopped up quickly with filter paper, weighed, finely chopped and suspended in a phosphate buffer (10 mL of sodium phosphate buffer 0.1 M, pH 7.5, per gram of wet tissue) for homogenization (30 strokes using a Potter-Elvehjem equipment) at 5 ºC.

The brain AChE activity was measured in the homogenates at room temperature (25-28 °C) according to [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF] with some modifications. The final concentrations in the enzyme assay medium were: dithio-bisnitrobenzoic acid (DTNB) 0.25 mM, TRITON X-100 0.1% and substrate 1 mM in phosphate buffer 0.1 mM, pH 7.5. The substrate was acetylthiocholine iodide (AceScol) and, when specified, replaced by propionyl (ProScol) or butyrylthiocholine iodides (ButScol) in the same concentration. The extinction coefficient of the reaction product (thionitrobenzoate ion) was 14,150 M -1 cm -1 in the spectrophotometer UV-160A (Shimadzu) at 412 nm. The activities are expressed in Units (1U = 1 µmol of product formed per min) per gram of wet tissue or per gram of total proteins (specific activity).

Five replicated assays carried out with each brain homogenate produced a coefficient of variation lower than 5%. The total proteins were determined as described by [START_REF] Peterson | A simplification of protein assay method of Lowry et al. which is more generally applicable[END_REF], with no precipitation step, using bovine serum albumin as standard.

The soluble AChE was determined in supernatants obtained by diluting the homogenates with phosphate buffer to 500 U/mL and centrifuging (30,000 x g/30 min/4 °C). The activity of AChE remaining in the supernatant was considered as soluble AChE and expressed as percentage (%) of the total activity. The pellets of these centrifugations were suspended in phosphate buffer with 0.1% of TRITON X-100, homogenized as described and centrifuged (100,000 x g/90 min/4 °C). The supernatants were collected and pooled by species for Michaelis-Menten constant (K M ) and IC50 determinations. The K M was calculated with non-linear regressions (Graph Pad Software, Inc.) with acetylthiocholine from 0.1 to 2 mM. The IC50-30min for methyl paraoxon was evaluated by the inhibition kinetic constants according to [START_REF] Johnson | Species-related differences in the inhibition of brain acetylcholinesterase by paraoxon and malaoxon[END_REF] and [START_REF] Kemp | Molecular determinants of species-selective inhibition of brain acetylcholinesterase[END_REF].

Phylogenetic correlations of the brain AChE specific activity among the collected species were assessed by hierarchic cluster analysis [START_REF] De Ghett | Hierarchical cluster analysis[END_REF]. The non-parametric cluster analysis of brain AChE specific activity and the Mann-Whitney test (U test) with the differences among the groups were carried out with Statistica 5.1 (Statsoft Inc.).

Results

The properties of the seawater sampled are shown in Table 2, except for station F where no samples were collected. Overall, 133 fishes were collected and classified into 11 families and 20 species (Table 3). The level of cerebral AChE in these species varied from 145 to 530 U/g of protein. The final results were normalized to total protein to compensate for water loss during storage. The soluble AChE varied from 21 to 42% of total activity calculated in the homogenates (Table 4).

The IC50-30 min for methyl paraoxon varied from 455 to 3,339 nM and the Michaelis-Menten constant (K M ) for acetylthiocholine varied from 104 to 291 µM for some of the species collected. The best substrate for the insoluble cerebral AChE was acetylthiocholine. Using propionyl or butyrylthiocholine values of 13 to 39% and of 1 to 16%, respectively, were obtained in relation to the results for acetylthiocholine (P% and B% in Table 5).

Figure 2 shows the result of a cluster analysis of the collected species which were separated in two groups at the level of super-order.

Discussion

Brain AChE specific activity was not influenced by the seawater physicochemical parameters. Variations of physicochemical parameters were very small and even in station B, where the greatest variations were observed, there was no correlation between the physicochemical parameters and AChE activity (Table 2). Similarly, [START_REF] Olson | Effects of water pollutants and other chemicals on fish acetylcholinesterase (In vitro)[END_REF] studied the effect of many chemicals in muscle AChE activity in fathead minnow (Pimephales promelas) and found that nitrate above 0.01 M caused a slight inhibition of the enzyme while phosphate and ammonium had no effect, even in concentrations above 0,01 M. In the present work, the concentrations of nitrate, phosphate and ammonium did not exceed 0.01 M in any water sample analyzed. However, brain AChE activity levels varied according to fish length. Pagrus pagrus, Porichthys porosissimus and Percophis brasiliensis showed the greatest correlation (Correlation Coefficient = -0.6333, -0.7470 and -0.80134, respectively), all negatives, indicating that older animals have less AChE in the brain. Reportedly, this ordinary variation in AChE activity relates to the biology and ecology of each species [START_REF] Bocquené | Characterization and assay conditions for use of AChE activity form several marine species in pollution monitoring[END_REF]. So, possible intraspecific variation in the brain AChE levels has to be evaluated to correctly interpret low results for brain AChE activity.

The variation among species, or interspecies AChE differences, might be ascribed to phylogeny according to [START_REF] Magnotti | Pesticide-sensitive fish muscle cholinesterases[END_REF]. The authors separated the species in two groups, one of high enzymatic activity and the other of low enzymatic activity. The group with the highest activities included families of the super-order Acanthopterygii. This super-order includes the more developed species. Our results were similar. A hierarchic cluster analysis (Figure 2) made with the brain AChE specific activities of the collected fish separated them into two groups: one with two species with the lowest brain AChE specific activity which belong to super-order Paracanthopterygii and other including the rest of the samples. The species in this latter group, except Genidens genidens, belong to super-order Acanthopterygii, with the highest brain AChE levels. Another two species, with only one sample, were not included in this analysis (Table 3). The super-order Paracanthopterygii includes species of primitive descent, according to [START_REF] Nelson | Fishes of the World[END_REF]. Our results confirm a possible linkage between the brain AChE activity levels and the phylogenetic origin of the fish.

The low soluble AChE activity found in brain of the fishes we studied (21 to 44%) support the hypothesis that the principal activity of this enzyme in brain, like in muscle, is particulate [START_REF] Massoulié | Molecular and cellular biology of cholinesterases[END_REF]. Our results showed that in the majority of the species studied a centrifugation step discarded up to 60 to 70% of the AChE activity (Table 4). Although several results were published using soluble AChE only [START_REF] Bocquené | Characterization and assay conditions for use of AChE activity form several marine species in pollution monitoring[END_REF][START_REF] Magnotti | Pesticide-sensitive fish muscle cholinesterases[END_REF][START_REF] Boone | Biochemical factors contributing to toxicity differences among chlorpyrifos, parathion, and methyl parathion in mosquito fish (Gambusia Affinis)[END_REF]Sturn, da Silva de Assis, & Hansen, 1999;[START_REF] Flammarion | The measurement of cholinesterase activities as a biomarker in chub (Leuciscus Cephalus): the fish length should not be ignored[END_REF][START_REF] Varò | Effect of Dichlorvos on Cholinesterase Activity of the European Sea Bass (Dicentrarchus Labrax)[END_REF], our findings reinforce the importance of using an AChE assay method without including a prior centrifugation step to discard pellets. So, in this report the brain AChE activity was assayed directly in the homogenates guaranteeing the detection of both enzyme activities (soluble and particulate).

The sensitivity of particulate brain AChE to methyl paraoxon varied from species to species (Table 5) and all brain AChE activities from the Neotropical fishes we studied were less sensitive to methyl paraoxon than AChE from other animals (Table 6).

Moreover, less sensitive fish species, like Merluccius hubsi and Percophis brasiliensis (Table 5) exhibited low brain AChE levels (Table 4). The Correlation Coefficient between brain AChE sensitivity (IC50-30min) and brain AChE levels (AChE) for some species (Table 4) was -0.75423. These results indicate that by selecting a sentinel species with low AChE activity levels one might risk biomarker's efficiency by selecting a species also with less sensitivity to the toxic substances.

The amount of enzyme activity is the common parameter when brain AChE is used as biomarker to pesticide exposure. A 50% reduction in specific activity in regard to non-exposed individuals suggests that these toxics are present in the environment (Burgeot, Bocquéné, Porte, Dimeet, Santella, Garcia de la Parra, [START_REF] Burgeot | Bioindicators of pollutant exposure in the Northwestern Mediterranean Sea[END_REF][START_REF] Kozlovskaya | Brain acethylcholinesterase and backbone collagen in fish intoxicated with with organophosphate pesticides[END_REF]. [START_REF] Bocquené | Characterization and assay conditions for use of AChE activity form several marine species in pollution monitoring[END_REF], working with several species of marine animals, suggested the utilization of the fish Pleuronectes platessa and the crustacean Palaemon serratus which, in comparing with others marine organisms, had the highest AChE specific activities. Besides, these species were the most sensitive organisms to the inhibitors, increasing the biomarker's efficiency.

For the Brazilian fishes studied, the sensibility to the pesticide was higher for enzymes with high K M or with low affinity for the natural substrate. The efficiency of hydrolysis of substrates with crescent alkyl chain also demonstrated that the more sensitive cerebral AChE is less specific for the natural substrate, as shown by the high values for P% and B% and the high K M for acetylthiocholine (Table 5). Just the inverse was observed by [START_REF] Kemp | Molecular determinants of species-selective inhibition of brain acetylcholinesterase[END_REF] for other animals. These authors suggested that the enzyme catalytic center should be more open in more sensitive animals, making substrate and inhibitor catalysis easier. They worked with widely different groups of animals: mammals, birds and fish, but our results, just for fish, do not encompass this latter broad grouping. These results indicate that the sensitivity to the pesticides and the specificity for the substrate can involve different mechanisms in different species.

Temperature can influence the K M and IC50 for the brain AChE in rat with little influence in changing the kinetic constants for this enzyme in channel catfish [START_REF] Carr | Kinetic analysis of the in vitro inhibition, aging, and reactivation of brain acetylcholinesterase from rat and channel catfish by paraoxon and chlorpyrifos-oxon[END_REF], which substantiate the concept that the higher resistance of AChE to organophosphate pesticide in some fishes can involve sites other than those involved in natural substrate and pesticide catalysis.

Conclusion

In conclusion, the brain AChE levels and its sensitivity to organophosphorus in fishes of the Rio de Janeiro coast are quite variable. Further in vitro and in vivo studies on the properties of the brain AChE present in each fish species will make it easier to choose marine sentinel species as a biomarker. However, our results have shown that a sentinel Neotropical fish species should have the highest brain AChE level among the more sensitive ones. The geographic position of the stations A-E were determined by GPS and station F was from a navigation map. The number in parenthesis represents the order of these species in relation to their AChE specific activity shown in the Table 4.

  Fig 1

Table 1

 1 Specifications of oceanographic expeditions (letters) and littoral sampling sites (numbers)

	organized by expedition dates
	Station	Date	City	Longitude -Latitude	Depth (m)
	A1	06/17/97 Arraial do Cabo	22° 58.690' S -042° 05.660' W	44.0
	A2	06/17/97		23° 00.910' S -042° 05.970' W	60.0
	B1	06/30/97 Macaé		22° 21.769' S -041° 43.630' W	7.5
	B2	07/01/97		22° 24.642' S -041° 38.103' W	20.0
	B3	07/02/97		22° 26.273' S -041° 46.680' W	31.0
	B4	07/03/97		22° 26.145' S -041° 40.000' W	30.0
	C1	08/20/97 Arraial do Cabo	22° 58.260' S -042° 03.560' W	30.0
	C2	08/20/97		22° 58.690' S -042° 05.660' W	45.0
	C3	08/20/97		23° 00.910' S -042° 05.970' W	60.0
	D1	08/21/97 Cabo Frio	22° 55.103' S -042° 00.642' W	30.0
	D2	08/21/97		22° 56.738' S -041° 58.523' W	45.0
	D3	08/21/97		23° 00.102' S -041° 53.091' W	60.0
	E1	09/29/97 Guanabara Bay	23° 05.189' S -043° 08.910' W	48.8
	E2	09/30/97 Guaratiba	23° 07.012' S -043° 32.310' W	45.0
	E3	10/03/97 Ilha Grande	23° 08.160' S -044° 23.480' W	29.0
	F1	08/24/98 Sepetiba Bay	23° 00.000' S -043° 45.000' W	6.0
	F2	03/28/99		23° 00.000' S -043° 45.000' W	6.0

Table 2

 2 Physical-chemical parameters of seawater in sampling sites

	Stations *	Temperature Salinity Oxygen °C ppm mL/L	PO 4	Nutrients (µ µ µ µM) NO 3	NH 3
	A1	15.64	35.66	4.58	0.63	6.82	1.02
	A2	15.54	35.74	4.56	0.67	7.26	1.05
	B1	22.50	36.16	6.30	0.46	0.07	1.17
	B2	20.92	36.36	5.72	0.57	1.55	1.96
	B3	21.86	36.22	5.89	0.34	0.38	0.86
	B4	19.80	36.37	5.56	0.57	3.94	1.11
	C1	15.88	35.62	4.32	0.70	8.22	0.89
	C2	15.68	35.58	4.60	0.65	8.14	1.06
	C3	15.54	35.56	4.20	0.65	8.39	1.10
	D1	17.48	35.85	4.96	0.67	7.18	1.22
	D2	17.60	35.85	4.78	0.64	6.46	1.53
	D3	16.92	35.75	4.84	0.66	7.79	1.07
	E1	21.84	nd	4.99	0.66	3.73	1.20
	E2	17.28	nd	4.36	0.54	3.56	1.09
	E3	21.12	nd	4.52	0.33	0.12	0.99
	nd -not determined

* Seawater was not collected in expedition F

Table 3

 3 Taxonomic and distribution of the studied fishes Brain AChE specific activities in the studied species (in brackets the number of animals tested)* the values are expressed as mean ± standard deviation of individual replicated assays in the brain of each collected fish.

	Family	Species	Common names	N Stations
	Ariidae	Genidens genidens	bagre-guri	6 F1 F2
	Batrachoididae Porichthys porosissimos	mamangá	7 B3 C2 C3 D3 E1 E2
	Bothidae	Paralichthys patagonicus	linguado	6 C1 C2 C3 D2 E2
	Merluccidae	Merluccius rubbsi	merluza	12 A1 A2 C2 C3
	Mullidae	Mullus argentinae	trilha	4 A2 E2
	Percophidae	Percophis brasiliensis	tira-vira	6 A1 A2 C2 E2
	Pomadasydae	Ortopristis ruber	corcoroca	4 E3
	Pomadasydae	Haemulon steindachneri	corcoroca-boca-larga	6 F1 F2
	Pomatomidae	Pomatomus saltatrix	enchova	1 D3
	Sciaenidae	Cynoscion striatus	pescadinha	8 A1 A2 D2
	Sciaenidae	Macrodon ancylodon	pescada-foguete	7 B3 B4 E3
	Sciaenidae	Umbrina canosai	castanha	5 A1 D1
	Sciaenidae	Stellifer rastrifer	cangoá	10 B1 E3
	Sciaenidae	Micropogonias furnieri	corvina	3 A1 D2
	Sciaenidae	Paralonchurus brasiliensis maria-luíza	14 B1 B2 B3
	Sciaenidae	Cynoscion jamaicensis	goete	3 B3 B4
	Sciaenidae	Menticirrhus americanus	papa-terra	8 B4 E3 F1 F2
	Sciaenidae	Ctenociaena gracilicirrhus cangauá	1 D2
	Serranidae	Dules auriga	mariquita	12 B1 C1 D2 E1
	Sparidae	Pagrus pagrus	pargo-róseo	10 A1 A2 D3 E1
			Total	133

***common names, number of fish collected and catch localisation are shown in Table

3

; 1U = 1 µ mol of product / min

Table 5

 5 Brain AChE kinetic characteristics of species collected in this work

	Species	IC50-30min	K M	P%	B%	AChE
	Genidens genidens	455	236	39	3	376
	Paralomchurus brasiliensis	468	228	32	16	389
	Haemulon steindachneri	1035	273	18	2	443
	Pagrus pagrus	1087	115	15	1	392
	Menticirrhus americanus	1579	291	20	1	435
	Cynoscion striatus	1595	206	17	1	297
	Dules auriga	1624	164	18	2	427
	Merluccius hubbsi	3259	107	28	nd	186
	Percophis brasiliensis	3339	104	13	nd	270
	nd -not detected
	IC50-30min -Pesticide concentration which inhibits 50% of AChE activity after
	30 min of incubation expressed in nanomol/liter (nM)
	K M -Michaellis-Menten constant with AceScol (µM)
	P% [(activity with ProScol/activity with AceScol) * 100] (%)
	B% [(activity with ButScol/activity with AceScol) * 100] (%)
	AChE -Specific activity of AChE in the brain (U/g protein, from Table 4)

Table 6

 6 Brain AChE sensitivity to organophosphates in various animals Pesticide concentration which inhibit 50% of AChE activity after 30 min of "in vitro" incubation expressed in nanomol/liter (nM) Sampling sites of the expeditions (A to F) in the Rio de Janeiro state coast Diagram of the hierarchic clusters analysis (Statistica 5.0 A , StatSoft, Inc.) for brain AChE activity of fish collected (simple linkage distance). (1) Porichthys porosissimos; (2) Merluccius rubbsi; (3) Percophis brasiliensis; (4) Cynoscion striatus; (5) Macrodon ancylodon; (6) Mullus argentinae; (7) Umbrina canosai;

	Organism	Pesticide	IC50-30min (nM)	Reference
	rat (37°C)	Ethyl paraoxon	13	Johnson & Wallace, 1987
		Methyl paraoxon	29	Kemp & Wallace, 1990
		Ethyl paraoxon	27	Carr & Chambers, 1996
	hem (37°C)	Ethyl paraoxon	37	Kemp & Wallace, 1990
	fish (Limanda limanda)	Ethyl paraoxon	312	Sturn et al., 1999
	fish (Serranus cabrilla)	Ethyl paraoxon	1,925	Sturn et al., 1999
	fish (rainbow trout at 11°C) Methyl paraoxon	3,310	Johnson & Wallace, 1987
	IC50 -

(8) Stellifer rastrifer; (9) Micropogonias furnieri; (10) Genidens genidens; (11) Paralonchurus brasiliensis; (12) Cynoscion jamaicensis; (13) Pagrus pagrus; (14) Ortopristis ruber; (15) Dules auriga; (16) Haemulon steindachneri; (17) Menticirrhus americanus; (19) Paralichthys patagonicus. The species Pomatomus saltador (18) and Ctenociaena gracilicirrhus (20) were not included because only one sample was collected.