ToxSci Advance Access originally published online on February 20, 2007
Toxicological Sciences 2007 97(1):81-93; doi:10.1093/toxsci/kfm022
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Correlation between Protein Accumulation Profiles and Conventional Toxicological Findings Using a Model Antiandrogenic Compound, Flutamide
* Bayer CropScience, Research Toxicology, Sophia-Antipolis, France
Faculté de Médecine Lyon Sud, INSERM U407, Oullins, France
1 To whom correspondence should be addressed at Bayer CropScience, 355 rue Dostoïevski, 06903 Sophia-Antipolis, France. Fax: 33 (0)4-93-95-84-54. E-mail: remi.bars{at}bayercropscience.com.
Received December 11, 2006; accepted February 11, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In conventional rodent toxicity studies the characterization of the adverse effects of a chemical relies primarily on gravimetric, and histopathological data. The aim of this study was to evaluate if the use of two-dimensional gel electrophoresis could generate protein accumulation profiles, which were in accordance with conventional toxicological findings by investigating a model antiandrogen, flutamide (FM), whose toxic effects, as measured using standard approaches, are well characterized. Male Sprague-Dawley rats were orally exposed to FM (0, 6, 30, and 150 mg/kg/day) for 28 days. The expected inhibition of androgen-dependent tissue stimulation, increased luteinizing hormone and testosterone plasma levels, and Leydig cell hyperplasia were observed. Changes in testicular protein accumulation profiles were evaluated in rats exposed to 150 mg/kg/day FM. Several proteins involved in steroidogenesis (e.g., StAR, ApoE, Hmgcs1, Idi1), cell cycle, and cancer (e.g., Ddx1, Hspd1) were modulated by FM, and these data provided molecular evidence for the hormonal and testicular histopathology changes recorded. Changes in proteins associated with spermatogenesis were also recorded, and these are discussed within the context of the testicular phenotype observed following FM treatment (i.e., normal spermatogenesis but Leydig cell hyperplasia). Overall, our data indicate that the combination of conventional toxicology measurements with omic observations has the potential to improve our global understanding of the toxicity of a compound.
Key Words: antiandrogen; flutamide; testis; proteomics; two-dimensional gel electrophoresis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In conventional toxicity studies, the identification of male reproductive toxicity relies on the measurement of weight changes in the reproductive tract (e.g., testis, prostate, and epididymis), on macroscopic and histological findings, and/or changes in hormone levels or sperm parameters (Andrews et al., 2001
; Okahara et al., 2000). Based on these phenotypic changes, the generated toxicity profiles correspond to the consequences of compound exposure. However, to understand the underlying cause of the toxicity and the molecular targets involved, more refined approaches are required. Previously, this would require the use of such techniques as immunohistochemistry, western blotting, qPCR analysis of specific genes or proteins, and the use of many individual studies, each designed to investigate one particular biochemical pathway or a specific enzyme. However, with recent advances in technology, we now have the opportunity to take a more comprehensive approach.
For several years, new "omics" tools, such as genomics, proteomics, and metabonomics, have emerged, which have the potential to provide new insights into toxicological studies. With these tools, we can now explore the global effect of a chemical at the molecular level and we have the ability to identify the molecular events (e.g., perturbation of biological pathways) that lead to the overt phenotypic changes (Leighton, 2005; Merrick and Bruno, 2004; Rockett, 2001; Wetmore and Merrick, 2004). Moreover, correlations between molecular events and the resultant phenotypic changes have the potential to provide very compelling and robust data concerning the mode of action of a chemical. In addition, such data could lead to the identification of biomarkers pertinent to the toxic effects.
The application of proteomics to toxicology is in its infancy (Wetmore and Merrick, 2004). For example, previous studies performed on the testis have been mainly focused on the characterization of proteins expressed in spermatogenic cells using two-dimensional gel electrophoresis (2-DE) (Chen et al., 2001; Com et al., 2003; Cossio et al., 1995, 1997; Flickinger et al., 2001; McKinnell et al., 1995), and only in a few cases have these tools been used to investigate testicular toxicity (Witzmann et al., 2003; Yamamoto et al., 2005). Thus, the aim of the present study was to evaluate if 2-DE would be able to generate protein accumulation profiles, which were in accordance with conventional toxicological findings (e.g., organ weight changes, histopathological findings, and/or hormone-level changes) recorded in the rat following compound exposure. To achieve this, we used a model antiandrogenic compound, flutamide (FM), whose effects in the rat testis have been well described in the literature. FM is a nonsteroidal antiandrogen, which is able to bind to the androgen receptor (AR); consequently, it can disturb the actions of endogenous androgens and can, therefore, give rise to reproductive organ toxicity (Peets et al., 1974; Simard et al., 1986; Viguier-Martinez et al., 1983). Although the toxic effects of FM are well characterized using conventional toxicity end points (Andrews et al., 2001; Kunimatsu et al., 2004; Okahara et al., 2000; Toyoda et al., 2000), little is known concerning changes occurring at the molecular level in the testes of FM-treated rats (Kubota et al., 2003; Ohsako et al., 2003). Furthermore, only very few studies have reported proteomic analyses with 2-DE on the antiandrogen FM (Cayatte et al., 2006; Pitkanen-Arsiola et al., 2006; Whitaker et al., 2004; Zhang et al., 2004). In accordance with a study duration typically used for compound evaluation in toxicology, male rats were treated daily for 28 days at various doses of FM (6, 30, and 150 mg/kg/day) by oral gavage to establish dose-response curves for the phenotypic changes (i.e., tissue weight changes, histopathological and hormonal changes). We then performed, for the first time, a global protein expression analysis on testicular tissue from control and treated rats using 2-DE. However, although 2-DE was performed on testicular samples from all treated animals, the omic work was mainly focused on testicular protein accumulation modulations detected at the high-dose level (150 mg/kg/day) FM to allow the interpretation of the molecular data and their correlation with the phenotypic changes.
Indeed, we have been able to detect changes at the molecular level (i.e., biological pathways affected), which are in accordance with the histopathological and hormonal changes observed in treated animals. In addition, the data generated in this study provided indications as to why we, as well as other investigators (Andrews et al., 2001; Kunimatsu et al., 2004; Toyoda et al., 2000), have been unable to detect any apparent histopathological changes on spermatogenesis in the testis following a 28-day exposure to FM.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals and housing.
The study was performed in accordance with The Guide for the Care and Use of Laboratory Animals (Council, 1996
). Male Sprague-Dawley (Ico:OFA-SD) rats (4 weeks of age on arrival) were purchased from Charles River Laboratories (St Germain-sur-l'Arbresle, France) and were acclimatized for 16 days. Each animal was individually housed in a stainless steel wire mesh cage under controlled environmental conditions (20°C-24°C, 40–70% relative humidity) with a 12-h light/dark cycle. Drinking water and pelleted rodent diet (Scientific Animal Food and Engineering, Epinay-sur-Orge, France) were available ad libitum. Prior to treatment, rats, judged to be in good health, were assigned to a group by the stratified randomization method based upon the body weight on the day before the initiation of administration, so that there was no significant variation in mean body weights among the groups.
Dosing and experimental design.
FM (CAS no. 034K1459; Sigma, St Quentin Fallavier, France) was administered in suspension to rats (6 weeks old, six per group) orally by gavage at a daily dose of 0 (control), 6, 30, or 150 mg/kg body weight, for 28 days, using a dose volume of 5 ml/kg body weight. Methylcellulose in sterilized water (0.5% wt/vol) was used as the vehicle for all FM dosing solutions. Clinical observations were performed daily, and body weights and physical examinations were recorded weekly. Twenty-four hours after the last dose, all animals were sacrificed by isoflurane (T.E.M, Lormont, France) inhalation followed by exsanguination under deep anesthesia. Terminal blood samples were collected from each animal and were placed into tubes containing lithium heparin. After centrifugation, plasma samples were removed and stored at – 80°C until hormone analysis. At necropsy, the following organs were excised from each rat, trimmed free of fat and connective tissue, and weighed: brain, kidney, liver, prostate (ventral and dorsolateral), testes, seminal vesicles, and epididymides. All paired organs were weighed together with the exception of the testes and epididymides, which were weighed separately. For each left testis, the capsule (tunica albuginea) was removed after which, the testis was cut into three equal parts and each resulting fraction was flash-frozen in liquid nitrogen and stored at – 80°C.
Histopathology.
The right testis was fixed for 72 h in 10% neutral-buffered formalin fixative (for possible further analyses by in situ hybridization). Paraffin-embedded tissue was prepared, sectioned at 
5 µm, and stained with hematoxylin (Sigma, St Quentin Fallavier, France) and eosin (Merck, Fontenay-sous-Bois, France) for histological examination. The tissue sections were examined under light microscopy. The severity of Leydig cell hyperplasia was graded from minimal to marked as follows—minimal (grade 1): an increase of Leydig cells within the interstitial space; slight (grade 2): an increase of Leydig cells with complete replacement of the interstitial space; moderate (grade 3): an increase of Leydig cells with complete replacement of the interstitial space and presence of bridging strands of Leydig cells surrounding the seminiferous tubules (one or two cell layers); and marked (grade 4): an increase of Leydig cells with complete replacement of the interstitial space and presence of bridging strands of Leydig cells surrounding the seminiferous tubules (more than two cell layers). Effects on spermatogenesis were also evaluated.
Hormone measurements.
Testosterone and luteinizing hormone (LH) levels were determined on individual plasma samples using specific radioimmunoassay kits (Beckman Coulter, Villepinte, France for testosterone and Amersham, Orsay, France for LH).
Two-dimensional gel electrophoresis.
Proteomic analysis was performed using total testicular proteins samples isolated from the left testis of individual animals, from all groups. Reagents and chemicals were mainly from Sigma-Aldrich-Fluka (St Quentin Fallavier, France). A frozen testicular sample from each animal was homogenized in lysis buffer (7M urea, 2M thiourea, 4% 3-[(3-cholamidopropyl)dimethylamonio]-1-propanesulfonate (CHAPS) (wt/vol), 0.24% Triton X-100 (vol/vol), 20mM spermine, and 50mM dithiothreitol) using a Dounce homogenizer. After incubating for 1 h at room temperature, 100mM iodoacetamide was added to each sample, and the mixtures were incubated for a further 3 h at room temperature in the dark. After centrifugation, the protein concentration of each resulting supernatant, diluted to 1/6, was determined using the modified Bradford protein assay (Ramagli and Rodriguez, 1985) using bovine serum albumin diluted in lysis buffer, as the standard. The protein concentration of each sample was then adjusted to 4 mg/ml prior to being aliquoted and stored at – 80°C.
Just prior to 2-DE, each testicular protein extract was diluted to 1 mg/ml using a rehydration buffer comprising 7M urea, 2M thiourea, 4% CHAPS, 0.24% Triton X-100, 0.6% bio-lyte, pH 3–10, ampholyte, 0.3% bio-lyte, pH 5–8, ampholyte (bio-lyte ampholyte 40%, Bio-Rad, Marnes-la-Coquette, France), 50 mM dithiothreitol, and 0.001% bromophenol blue as a tracking dye. The isoelectric focusing (IEF) step was carried out using a Protean IEF Cell (Bio-Rad). Precast immobilized pH gradient (IPG) strips (readystrip IPG strips pH range 5–8, 17 cm long, Bio-Rad) were rehydrated with 400 µl (400 µg) of total testis protein and incubated for 22 h at 20°C (passive sample loading). Then, proteins were focused successively for 2 h at 50 V, 15 min at 250 V, followed by a slow (5 h) voltage ramping to 10,000 V, and focusing was continued at 10,000 V up to 80,000 Vh. All IEF steps were carried out at 20°C under low-viscosity oil. After the first-dimension IEF, the IPG strips were incubated at room temperature for 30 min in an equilibration buffer (0.375M Tris-HCl [pH 8.8], 6M urea, 20% glycerol, 4% sodium dodecyl sulfate [SDS]) containing 65mM dithiothreitol. A second equilibration step was carried out for 40 min under the same conditions, except that dithiothreitol was replaced by 135mM iodoacetamide and 0.001% bromophenol blue was added as a tracking dye. The equilibrated strips were then loaded onto 10% SDS-polyacrylamide gels (PAGE), with a stacking gel of 2 cm of 4% SDS-PAGE placed on the top. IPG strips were sealed with 1% low melting point agarose to ensure good contact between the IPG strips and the gel. The second dimension of 2-DE was carried out using the Ettan Daltsix Electrophoresis Unit (Amersham, Orsay, France) connected to a PowerPac 1000 power supply (Bio-Rad). The electrophoresis was performed at 10°C with Tris-glycine buffer (25mM Trizma base, 192mM glycine, and 0.1% SDS) at a constant power of 5 W per gel for 30 min followed by 12.5 W per gel until the tracking dye reached the bottom of the gel. After separation, proteins were visualized by staining the gels with colloidal Coomassie blue stain as recommended by the manufacturer (Blue SafeStain Invitrogen, Cergy Pontoise, France).
Image acquisition and analysis.
Gels were digitalized using the GS-800 calibrated densitometer (Bio-Rad), high-resolution scanner. The protein pattern of each gel (six gels per dose level; one gel per testis sample) was compared using the PD-Quest image analysis system (version 7.2, Bio-Rad). Briefly, after automatic spot detection, the images were edited manually, for example, adding, splitting, and removing spots. Automatic matching of spots on each gel was then performed. For each dose, a match set consisting of 12 images, six for control and six for treated rat testis gels was prepared. This resulted in the creation of three independent match sets (one for each dose level). For each match set, one control gel was selected (based on gel and spot quality) as the master gel against which all remaining gels were matched. For each gel, the intensity of each protein spot was normalized to the total intensity of the entire gel. The accumulation level of each protein was quantified by determining the intensity of each spot. Then the six individual gels from each treatment group were combined into groups for statistical analysis, and differences in protein accumulation between control group and treated group, for each dose level, were assessed. A change in spot intensity was considered to be an indication of the up- or down-accumulation of protein. Thus, protein spots with a statistically significant change in protein accumulation level were obtained for each match set. A number of these protein spots were selected for protein identification based on the quantity (intensity) of the protein spot (i.e., ability to see the spot on the gel) as well the quality of the spot on the gel (i.e., no streaking or overlapping with neighboring spots). Automatic determination of the experimental relative molecular mass (Mr) and isoelectric point (pI) values for each selected protein spots was performed by PD-Quest using standard protein markers (Bio-Rad).
In-gel tryptic digestion and protein identification by mass spectrometry.
The protein spots of interest were manually excised from the 2-DE gel. Each protein sample was further processed by enzymatic digestion with trypsin to generate peptides fragment (Cayatte et al., 2006). Mass spectra were recorded with a matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) spectrometer (Voyager DE-PRO, PerSeptive Biosystems, Framingham, MA) set in positive reflectron mode. Peptide mass profiles obtained by MALDI-MS were analyzed using Protein Prospector. Peptide masses were compared with the theoretical masses derived from the sequence contained in SWISS-PROT/TrEMBL databases or deduced from the nrNCBI Data Bank. The search parameters were set as follows: cysteines as carbamidomethyl derivative; allowed peptide mass error, 50–100 ppm; at least four peptide mass hits required for protein match; up to one missed cleavage; and methionine-oxidized form. Restrictions were placed neither on species of origin, pI, nor on protein mass range.
To confirm peptide mass fingerprinting results and when peptide mass fingerprinting could not unambiguously identify a protein, samples were analyzed by nano ESI-MS/MS (static infusion) on a LCQDECA XP-PRO mass spectrometer (ThermoQuest, San Jose, CA). Nano ES capillaries (ES 381, PROXEON Biosystems, Odense, Denmark) were used as emitter (Cayatte et al., 2006).
Functional analysis.
Functional classification of proteins modulated by FM treatment among the different metabolic pathways and subcellular location was based on Gene Ontology using rat genome database (http://rgd.mcw.edu), Kyoto Encyclopedia of Genes and Genomes (KEGG) encyclopedia (http://www.genome.ad.jp/kegg/genes.html) and Ingenuity Pathways Analysis (IPA) (Ingenuity Systems, http://www.ingenuity.com). IPA mapped each modulated protein to its corresponding gene object (e.g., genes, mRNAs, and proteins) in the Ingenuity Pathways Knowledge Base (IPKB). These genes, called "focus gene" were overlaid onto a global molecular network developed from information contained in the IPKB and were used as the starting point for generating biological networks, based on their connectivity. The functional analysis of a network identified the biological functions and/or diseases that were most significant to the genes in the network. The network associated with biological functions and/or diseases in the IPKB was considered for the analysis. Genes mapped to biological networks available in the IPKB were ranked by score. The score is the probability that a collection of genes equal to or greater than the number in a network could be achieved by chance alone. A score of 3 indicates that there is a 1/1000 chance that the focus genes are in a network due to random chance. Therefore, scores of 3 or higher have a 99.9% confidence level of not being generated by random chance alone. This score, was used as the cutoff for identifying gene networks significantly affected by FM.
Statistical analyses.
Statistical analyses on body and organs weights and hormonal parameters were performed as previously described (Kennel et al., 2004). For proteomic analysis, the mean protein intensity of control group was compared to the mean protein intensity of each treated group using the Mann-Whitney test. Statistical tests were performed at p 
0.05 and p 0.01 of significance using PD-Quest software (version 7.2, Bio-Rad).
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RESULTS |
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Body and Sex Organ Weights
FM was not overtly toxic to rats at any dose level investigated. However, a significant decrease (p
0.01) in body weight gain was recorded for those animals treated at 150 mg/kg/day FM (Table 1). Although the testicular weight was not affected following FM treatment, a dose-related decrease in most of the sex organs and accessory tissues was recorded, with statistically significant differences observed for the seminal vesicles, ventral and dorsolateral prostate, and the epididymides (Table 1). As the tissue weights were significantly (p 0.05, p 0.01) decreased at doses where the body weight was not adversely affected, these atrophic effects were considered to be treatment related.
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Histopathology and Hormone Levels
A treatment-related change in the testicular interstitial space, identified as Leydig cell hyperplasia (Fig. 1), was recorded in all treated groups. Both the severity (minimal to marked) and incidence of this lesion increased in a dose-related manner (Table 2). No significant morphological differences were observed in the seminiferous tubules between control and treated animals indicating that spermatogenesis was apparently not affected by treatment.
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At sacrifice, dose-related increases in the mean plasma levels of LH and testosterone were recorded (Fig. 2). At 6 mg/kg/day FM treatment, only testosterone was significantly (p
0.01) affected (4.2-fold increased). Both testosterone and LH levels were significantly increased at 30 mg/kg/day FM, to 8.6-fold (p 0.01) and 3.1-fold (p 0.05), respectively, and at 150 mg/kg/day FM, to 12.3-fold (p 0.01) and 10.3-fold (p 0.01).
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Proteomic Analysis
Whole testicular protein extracts from individual control and FM-treated (6, 30, and 150 mg/kg/day) rats were analyzed by 2-DE. Approximately 1500 proteins spots were detected on each gel. A representative protein profile of rat testis treated with 150 mg/kg/day FM is shown in Figure 3.
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Approximately 100 proteins were found altered at the high-dose level of 150 mg/kg/day based on their p-value (p
0.05) of which the 44 most abundant proteins were selected for mass spectrometry protein identification (MALDI-ToF and/or ESI-MS/MS) (Fig. 3). For each of the identified proteins, the identification number on the gel, the accession number from SWISS-PROT/TrEMBL or nrNCBI, the protein and gene name, MOWSE scores, matched peptides and protein coverage from mass spectrometry analysis, cell type localization in rodents, and the mean normalized spot intensity ratio of treated to control, for each dose level, are presented in Table 3. For more than 97% of spots, protein sequence coverage of at least 15% was obtained. Moreover, in most cases, the experimental Mr and pI values from 2-DE gels were in agreement with the theoretical Mr and pI values of the identified proteins. Thus, a high degree of confidence in the identified protein spots was obtained. The magnitude of ratio changes ranged from 0.39 down-accumulation (alpha-1-antiproteinase, spot no. 2) to 6.32 up-accumulation (aldo-keto reductase 1, member B8, spot no. 40). Higher ratios were observed for one protein spot, retinal dehydrogenase (spot no. 43), as it was uniquely accumulated due to treatment. In addition, the majority of statistically significantly modulated protein spots at the highest dose level were up-accumulated in a dose-related manner as highlighted in Figure 4. Among the identified proteins, few (e.g., spot no.7, 17, or 28) were located in an unexpected position on the gel based on their Mr and pI theoretical values. Changes in Mr and pI are most probably attributed to posttranslational protein modifications, such as proteolytic cleavage, glycosylation, and phosphorylation. Furthermore, a number of different protein spots were identified as being the same protein. For example, spots nos. 23, 33, and 34 (Table 3) were identified as aldehyde dehydrogenase mitochondrial. These proteins could be degradation products or different isoforms of the same protein. Thus, from the original 44 spots selected for identification, a total of 39 individual proteins were identified.
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Functional Analysis
Functional classification, according to cellular processes and the subcellular location of the identified proteins, was performed using Gene Ontology with the Rat Genome Database (http://rgd.mcw.edu), KEGG encyclopedia (http://www.genome.ad.jp/kegg/genes.html), and IPA (Ingenuity Systems, http://www.ingenuity.com). IPA was able to map the 39 modulated proteins at the high-dose level of FM to its corresponding gene object (e.g., genes, mRNAs, and proteins) in the IPKB. Focus genes for 32 of the 39 modulated proteins were overlaid onto a global molecular network developed from information contained in the IPKB. Networks of these focus genes were then algorithmically generated based on their connectivity. Biological pathways were assigned to each network and were ranked according to the significance of that biological function to the network. Three of the six networks identified were found to be highly significant in that they had more of the identified proteins present than would be expected by chance (Table 4). Lipid metabolism (34% of the identified proteins), that is, those cellular processes leading to the production of the major testicular androgen testosterone, was identified as the main biological pathway affected. Proteins associated with this pathway included those involved in glycerolipid metabolism (aldose reductase, aldehyde dehydrogenase), in fatty acid metabolism (gonadotropin-regulated long-chain acyl-CoA synthetase; acyl-CoA dehydrogenase, long- and short-chain specific, mitochondrial; enoyl coenzyme A hydratase 1, peroxisomal; fatty acid–binding protein heart), in sterol biosynthesis (hydroxymethylglutaryl-CoA synthase, cytoplasmic; similar to acetyl-CoA transferase-like; isopentenyl-diphosphate delta-isomerase 1; steroidogenic acute regulatory protein [StAR]; apolipoprotein E [ApoE]) and in retinoic acid biosynthesis (retinal dehydrogenase, aldehyde dehydrogenase, mitochondrial). The second and third networks identified included proteins involved in carbohydrate metabolism and in cell cycle, cell death, and cancer. Figure 5A portrays the 39 modulated proteins at the high-dose level of FM according to their subcellular location. The majority of proteins affected by the FM treatment were cytoplasmic whereas few were nuclear or secretory.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The pharmacological and toxic effects of the antiandrogen FM on the reproductive tract have been well characterized and described in the literature. In the present study, gravimetric, histological, and hormonal data were consistent with previously reported data (Andrews et al., 2001
; Kunimatsu et al., 2004; Toyoda et al., 2000; Viguier-Martinez et al., 1983). More specifically, a dose-related inhibition of androgen-dependent tissue stimulation was recorded, as well as an increase in LH and testosterone plasma levels. In addition, histopathological observations in the testis showed Leydig cell hyperplasia in the absence of any visible effect on spermatogenesis, which is in agreement with previously reported histopathology and sperm analysis data following a 28-day FM treatment (Andrews et al., 2001; Toyoda et al., 2000). Thus, taken together, our results confirmed the expected antiandrogenic effect of FM on the male rat reproductive tract. Having confirmed, phenotypically, the antiandrogenic effects of FM, we then performed global proteomic analyses to investigate those protein accumulation modulations in the testis following FM treatment to examine if there was a correlation between the testicular histopathological findings and the changes in protein profiles detected in treated animals. We focused, in the first instance, on the effect of the high-dose level of FM in order to maximize the level of modulation of the testicular proteins affected by the treatment. As expected, the severity and incidence of testicular lesions observed were found at their highest level at 150 mg/kg/day FM.
The accumulation levels of more than 100 testicular proteins were affected by 150 mg/kg/day FM, of which 44 proteins were identified by MALDI-MS and/or ESI-MS/MS (Table 3). These proteins were classified according to their cellular process and subcellular location (Table 4 and Fig. 5), and most of these proteins were found to be involved in lipid metabolism, or in cell cycle and cancer.
Several proteins, specific to Leydig cells in rodent testis and involved in steroidogenesis, were found to be modulated by treatment (Table 3 and Fig. 6). For example, StAR (spot no. 17) and ApoE (spot no. 1), which are associated with cholesterol transport and distribution, were upregulated in a dose-response manner. The StAR protein is involved in the transport of cholesterol to the inner mitochondrial membrane (Luo et al., 1998), which is the first and rate-limiting step in steroid biosynthesis in the adrenals and gonads, and ApoE appears to play a major role in the regulation and redistribution of cholesterol between tissues or among different cells within a tissue (Fofana et al., 2000; Olson et al., 1994). In addition to these two key steroidogenic proteins, five other proteins involved in fatty acid metabolism were modulated by treatment: gonadotropin-regulated long-chain acyl-CoA synthetase (Grlacs, spot no. 14), acyl-CoA dehydrogenase, long-chain–specific mitochondrial (Acadl, spot no. 29), acyl-CoA dehydrogenase, short-chain–specific mitochondrial (Acads, spot no. 30), enoyl coenzyme A hydratase 1 peroxisomal (Ech1, spot no. 28), and fatty acid–binding protein heart (Fabp3, spot no. 9). Two additional proteins, also associated with steroidogenesis, were upregulated following FM treatment: hydroxymethylglutaryl-CoA synthetase cytoplasmic (Hmgcs1, spot no. 11), which belongs to the ketogenic pathway converting acetyl-CoA to HMG-CoA (a sterol biosynthesis precursor) (Mascaro et al., 2000) and isopentenyl-diphosphate delta-isomerase 1 (Idi1, spot no. 10), which is involved in a crucial activation step in the isoprenoid biosynthetic pathway (Barella et al., 2004).
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Several proteins involved in carbohydrate metabolism (Fig. 6) were also identified, and it is likely that these participate indirectly in steroid biosynthesis as a source of energy and steroid precursors. As testes have a large requirement for cholesterol for the production of testosterone, it is conceivable that the up-accumulation of all these proteins (associated with steroidogenesis and carbohydrate metabolism) could lead to an increase in the cholesterol pool available for steroid biosynthesis. Moreover, measurements of mRNA transcription level of four of the steroidogenesis-related proteins (StAR, Fabp3, Grlacs, Hmgcs1) (real-time RT-PCR) confirmed that gene expressions for some of these proteins were also consistently upregulated by FM treatment (data not shown). Overall, several proteins involved, in a direct or indirect manner, in the steroidogenesis metabolic pathway were affected by FM, thus providing evidence, at the molecular level, for the hormone-related changes observed following FM treatment, namely the increase in plasma testosterone level.
A second group of proteins affected following FM treatment was found to be involved in cell cycle and cancer. Included in this group were transcription regulators carbonic anhydrase 2 (Ca2, spots nos. 39 and 41) and lamin A (Lmna, spot no. 24), the translation regulator, DEAD (Asp-Glu-Ala-Asp) box polypeptide 1 (Ddx1, spot no. 44), chaperon proteins chaperonin 60 (Hspd1, spots nos. 3 and 4) and TNF receptor–associated protein 1 (Trap1, spot no. 20), as well as several potent antiproliferative proteins prohibitin (Phb, spot no. 7) and fatty acid–binding protein heart (Fabp3, spot no. 9). As all these proteins were upregulated due to FM treatment and correlated well with the observed histopathological lesions, that is, hyperplasia of the Leydig cells, they appear to be involved in cellular growth, proliferation, and differentiation processes.
Having established a good correlation between the phenotypic changes observed in the testis following FM exposure and the corresponding molecular events, we turned our attention to other modulated proteins, in the hope that we would gain even further insight into the mode of action of FM.
For example, some proteins modulated by FM exposure were associated with spermatogenesis. We hypothesized that the modulation of some of these proteins could explain the testicular phenotype observed following FM exposure, that is, a normal spermatogenetic process, despite the AR blockade and Leydig cell hyperplasia. We specifically focused on retinal dehydrogenase 1 (Aldh1a1) (spot no. 43) for several reasons. First, this enzyme is associated with the oxidation of different aldehydes, including all-trans retinal to retinoic acid in the rat testis (Cavazzini et al., 1996; Duester, 1996, 2000) and it is known that vitamin A (retinol) and its principal biologically active derivative, retinoic acid, are involved in the regulation of testicular functions in rodents (Livera et al., 2002). Second, published data have demonstrated that retinoids can also increase the transcription and translation of several proteins implicated in the testicular functions (Livera et al., 2002). Finally, retinoic acid has been shown to reinitiate spermatogenesis in vitamin A–deficient rats (van Pelt and de Rooij, 1991). Thus, in addition to an upregulation of Aldh1a1, we also identified a number of significantly modulated proteins that are regulated by retinoids, for example, StAR (spot no. 17), serotransferrin (Tf, spot no. 37), and cytochrome P450c17 (Cyp17a1) (Lefevre et al., 1994) (change observed by qPCR analysis—data not shown). Thus, based on our own observations, coupled with previously published data, it is conceivable that the normal spermatogenic process observed in the testes following 28-days FM treatment could be, at least partially, due to the increase in Aldh1a1 leading ultimately to an increase in retinoic acid concentration. However, further experiments are necessary to confirm this hypothesis.
Finally, some proteins modulated following FM exposure could not be related to the observed hormone-level changes or histopathological lesions in the testis. Thus, further investigations will be necessary to understand their roles and implications in the Leydig cell hyperplasia or other treatment-related changes not readily detected by histopathology.
Two-dimensional electrophoresis proved to be a reliable and powerful tool to evaluate molecular changes in rat testes following FM treatment. Indeed, data generated in this laboratory have indicated that similar testicular protein profiles can be obtained from similar experiments in rats treated with FM conducted 1 year apart, demonstrating the reproducibility of the data. However, this technique does have some limitations. For example, proteins with a high molecular weight or with pI values that fall at the extremities of the pH gradients (very acidic or basic proteins) are difficult to resolve on a gel. Furthermore, it is well known that proteins of low abundance or certain classes of proteins, such as integral membrane proteins or nuclear proteins, are underrepresented on classic 2-DE (Gorg et al., 2004; Rabilloud, 2002). In fact, our data generated by the IPA tool (Fig. 5A) indicated that the majority of proteins altered by FM treatment were cytoplasmic or secreted; very few or none were found in the nucleus or the membrane. Whereas the analysis of low-abundance proteins could probably be improved by fractionating the testis and purifying the various cell types before proteomic analysis (Ahmed and Rice, 2005), the nuclear and membrane proteins will be clearly difficult to analyze. However, the use of the IPA tool could allow us to overcome these limitations. Specifically, the identification of the altered protein spots, the analysis of networks, and the describing of functional relationships between gene products reported in literature could provide clues as to other gene products also altered by FM treatment, which cannot be detected by 2-DE. For example, Hspd1, StAR, Aldh1a1, Acat2 (acetyl-Coenzyme A acetyltransferase 2, spot no. 31) or Akr1b8 (aldo-keto reductase family 1, member B8, spot no. 40) were modulated following FM treatment and all interact, either directly or indirectly, with the transcription factors Jun or retinoic acid nuclear receptor RXR/RAR (Fig. 5B), which would themselves be difficult to detect by 2-DE. It would appear therefore that these transcription factors, as well as other poorly expressed gene products belonging to the same signaling cascade, could also be affected by FM treatment. However, this hypothesis remains to be tested using western blot or specific qPCR analyses. Nevertheless, the combination of protein profiles and IPA investigations may help to identify new genes altered by the FM treatment and so provide further insights into the toxic mode of action of FM.
In summary, we detected changes in testicular protein accumulation of rats treated with a high dose of FM, a known antiandrogenic compound. Alterations in the protein profiles showed that specific metabolic pathways were affected and that these molecular changes could be correlated with the hormone-level changes and the testicular histopathological changes following FM treatment. Thus, evidence for the testicular toxicity due to a high dose of FM was obtained at the molecular level as well as phenotypically, leading to an improved understanding of the global changes induced by FM in the testis.
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ACKNOWLEDGMENTS |
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The authors would like to thank Michel Samson, INSERM 638, Nice, for MS analysis; Laëtitia Elies for histopathological expertise; and Céline Katz for bioinformatic analysis. The first author was recipient of a fellowship co-sponsored by the "Association Nationale de la Recherche Technique" and Bayer CropScience.
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