Correlation between protein accumulation profiles and conventional toxicological findings using a model antiandrogenic compound, flutamide.

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.

[1]  John K Leighton,et al.  Application of Emerging Technologies in Toxicology and Safety Assessment: Regulatory Perspectives , 2005, International journal of toxicology.

[2]  H. Tanioka,et al.  Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats 5). Effects of repeated doses of flutamide for 2 and 4 weeks. , 2000, The Journal of toxicological sciences.

[3]  G. Livera,et al.  Regulation and perturbation of testicular functions by vitamin A. , 2002, Reproduction.

[4]  D. Hochstrasser,et al.  Spermatocytes and round spermatids of rat testis: Protein patterns , 1995, Electrophoresis.

[5]  G. Rimbach,et al.  Alpha-tocopherol affects androgen metabolism in male rat. , 2004, Annals of the New York Academy of Sciences.

[6]  K. Toyoda,et al.  Repeated dose (28 days) oral toxicity study of flutamide in rats, based on the draft protocol for the `Enhanced OECD Test Guideline 407' for screening for endocrine-disrupting chemicals , 2000, Archives of Toxicology.

[7]  A. Lefèvre,et al.  Regulation by retinoids of luteinizing hormone/chorionic gonadotropin receptor, cholesterol side-chain cleavage cytochrome P-450, 3β-hydroxysteroid dehydrogenase/Δ5−4-isomerase and 17α-hydroxylase/C17–20 lyase cytochrome P-450 messenger ribonucleic acid levels in the K9 mouse Leydig cell line , 1994, Molecular and Cellular Endocrinology.

[8]  B Alex Merrick,et al.  Genomic and proteomic profiling for biomarkers and signature profiles of toxicity. , 2004, Current opinion in molecular therapeutics.

[9]  J. Simard,et al.  Characteristics of interaction of the antiandrogen flutamide with the androgen receptor in various target tissues , 1986, Molecular and Cellular Endocrinology.

[10]  C. Pineau,et al.  New Insights into the Rat Spermatogonial Proteome , 2003, Molecular & Cellular Proteomics.

[11]  S. Ottonello,et al.  Retinoic acid synthesis in the somatic cells of rat seminiferous tubules. , 1996, Biochimica et biophysica acta.

[12]  R. Neri,et al.  On the mechanism of the anti-androgenic action of flutamide (alpha-alpha-alpha-trifluoro-2-methyl-4'-nitro-m-propionotoluidide) in the rat. , 1974, Endocrinology.

[13]  B. Zirkin,et al.  Leydig cell protein synthesis and steroidogenesis in response to acute stimulation by luteinizing hormone in rats. , 1998, Biology of reproduction.

[14]  C. Tohyama,et al.  Alterations of gene expression in adult male rat testis and pituitary shortly after subacute administration of the antiandrogen flutamide. , 2003, The Journal of reproduction and development.

[15]  L. Ramagli,et al.  Quantitation of microgram amounts of protein in two‐dimensional polyacrylamide gel electrophoresis sample buffer , 1985 .

[16]  H. Yamada,et al.  Protein expression analysis of rat testes induced testicular toxicity with several reproductive toxicants. , 2005, The Journal of toxicological sciences.

[17]  G. Rimbach,et al.  α‐Tocopherol Affects Androgen Metabolism in Male Rat , 2004 .

[18]  Genomic and proteomic profiling for biomarkers and signature profiles of toxicity. , 2004 .

[19]  Fu Gao,et al.  Nuclear matrix in developing rat spermatogenic cells , 2001, Molecular reproduction and development.

[20]  P. Andrews,et al.  Feasibility and potential gains of enhancing the subacute rat study protocol (OECD test guideline no. 407) by additional parameters selected to determine endocrine modulation. A pre-validation study to determine endocrine-mediated effects of the antiandrogenic drug flutamide , 2001, Archives of Toxicology.

[21]  R. Sharpe,et al.  Comparative analysis of proteins secreted in vitro by isolated seminiferous tubules from man and the rat. , 1995, International journal of andrology.

[22]  D. Hochstrasser,et al.  Spermatocytes and round spermatids of rat testis: The difference between in vivo and in vitro protein patterns , 1997, Electrophoresis.

[23]  A. LeBlanc,et al.  Estrogen and Androgen Protection of Human Neurons against Intracellular Amyloid β1-42 Toxicity through Heat Shock Protein 70 , 2004, The Journal of Neuroscience.

[24]  D. Rouquié,et al.  Protein Profiling of Rat Ventral Prostate following Chronic Finasteride Administration , 2006, Molecular & Cellular Proteomics.

[25]  S. Gamble,et al.  Androgen Receptor Is Targeted to Distinct Subcellular Compartments in Response to Different Therapeutic Antiandrogens , 2004, Clinical Cancer Research.

[26]  L. Olson,et al.  Immunolocalization of apolipoprotein E in the testis and epididymis of the rat. , 1994, Biology of reproduction.

[27]  S. Carreau,et al.  Evaluation of cholesteryl ester transfer in the seminiferous tubule cells of immature rats in vivo and in vitro. , 2000, Journal of reproduction and fertility.

[28]  R. Bars,et al.  Evaluation of the rodent Hershberger assay using three reference endocrine disrupters (androgen and antiandrogens). , 2004, Reproductive toxicology.

[29]  Y Ohno,et al.  Collaborative work to evaluate toxicity on male reproductive organs by repeated dose studies in rats--overview of the studies. , 2000, The Journal of toxicological sciences.

[30]  J. Rockett.  Genomic and proteomic techniques applied to reproductive biology , 2001, Genome Biology.

[31]  A. Görg,et al.  Current two‐dimensional electrophoresis technology for proteomics , 2004, Proteomics.

[32]  R. Neri,et al.  On the Mechanism of the Anti-androgenic Action ofFlutamide (α-α-α-Trifluoro-2-methyl-4'-nitro-m-propionotoluidide)in the Rat , 1974 .

[33]  N. Sherman,et al.  Outer Dense Fiber Proteins Are Dominant Postobstruction Autoantigens in Adult Lewis Rats1 , 2001, Biology of reproduction.

[34]  Nuzhat Ahmed,et al.  Strategies for revealing lower abundance proteins in two-dimensional protein maps. , 2005, Journal of chromatography. B, Analytical technologies in the biomedical and life sciences.

[35]  C. Tohyama,et al.  Effects of vinclozolin administration on sperm production and testosterone biosynthetic pathway in adult male rat. , 2003, The Journal of reproduction and development.

[36]  Barbara A Wetmore,et al.  Toxicoproteomics: proteomics applied to toxicology and pathology. , 2004, Toxicologic pathology.

[37]  Division on Earth.  Guide for the Care and Use of Laboratory Animals , 1996 .

[38]  C. Perreau,et al.  Endocrinological and histological changes induced by flutamide treatment on the hypothalamo-hypophyseal testicular axis of the adult male rat and their incidences on fertility. , 1983, Acta endocrinologica.

[39]  G. Duester.  Involvement of Alcohol Dehydrogenase, Short-Chain Dehydrogenase/Reductase, Aldehyde Dehydrogenase, and Cytochrome P450 in the Control of Retinoid Signaling by Activation of Retinoic Acid Synthesis† , 1996 .

[40]  F. Witzmann,et al.  Analysis of rat testicular protein expression following 91‐day exposure to JP‐8 jet fuel vapor , 2003, Proteomics.

[41]  T. Rabilloud.  Two‐dimensional gel electrophoresis in proteomics: Old, old fashioned, but it still climbs up the mountains , 2002, Proteomics.

[42]  G. Coetzee,et al.  Androgen and anti‐androgen treatment modulates androgen receptor activity and DJ‐1 stability , 2006, The Prostate.

[43]  G. Duester.  Families of retinoid dehydrogenases regulating vitamin A function: production of visual pigment and retinoic acid. , 2000, European journal of biochemistry.

[44]  T. Seki,et al.  Evaluation for reliability and feasibility of the draft protocol for the enhanced rat 28-day subacute study (OECD Guideline 407) using androgen antagonist flutamide. , 2004, Toxicology.

[45]  D. D. de Rooij,et al.  Retinoic acid is able to reinitiate spermatogenesis in vitamin A-deficient rats and high replicate doses support the full development of spermatogenic cells. , 1991, Endocrinology.