TY - JOUR
AU - Bars,, Rémi
AB - Abstract An important step in the safety assessment of chemicals for humans is to determine the no observed adverse effect level (NOAEL) in toxicity studies conducted in animal models. With the increasing use of molecular tools in toxicity studies, a question often posed is how a NOAEL derived from molecular data compares to a NOAEL established using standard methods. The objective of the present study was to address this question when considering testicular toxicity. To do this, we assessed the effects of the reference antiandrogen flutamide on rat testes in a standard 28-day toxicity study using doses of 0.04–150 mg/kg/day. At necropsy, blood samples were collected for testosterone measurements. The testes were collected for histopathological assessment as well as for the evaluation of gene expression changes using quantitative PCR analyses. Results showed that increases in plasma testosterone level and Leydig cell hyperplasia were detected from 6 mg/kg/day. An alteration in the level of accumulation of a selection of genes was also detected from 6 mg/kg/day. This was the case for genes functionally associated with the testicular lesion, such as lipid metabolism and cell death/cell proliferation, as well as for genes not functionally associated with the lesion. Contrary to the misgivings, these data show that, using a standard 28-day toxicity study and a well-characterized adverse effect, the NOAEL based on transcript changes is similar to the NOAELs based on testosterone levels and histopathological examination. antiandrogen, flutamide, Toxicogenomics, NOAEL, dose response The safety assessment of chemicals for humans relies on the determination of the no observed adverse effect levels (NOAELs) in toxicity studies using animal models and standard methods. However, as molecular tools develop, a question often posed is how a NOAEL derived from molecular data compares to a NOAEL established using standard methods (Cunningham et al., 2003; Freeman, 2004). Consequently, as part of our ongoing investigations into endocrine-related testicular toxicity (Friry-Santini et al., 2007), we took the opportunity to address this apprehension. Thus, with the objective of establishing NOAELs for testicular toxicity based on both standard and Toxicogenomics (OMICs) data generated in the same study, we conducted two independent dose-response studies in the rat using the reference antiandrogen, flutamide (FM) (Andrews et al., 2001; Friry-Santini et al., 2007; Kunimatsu et al., 2004; Tinwell et al., 2007; Toyoda et al., 2000). So as to be consistent with standard approaches, we conducted the 28-day repeat-dose oral toxicity studies based on the Organization for Economic Cooperation and Development test guideline 407. The dose levels of FM (0.04–150 mg/kg/day) were selected to allow a clear phenotypic anchoring of the molecular data at the top dose levels as well as discrimination between the phenotypic and the molecular changes at the low doses if such a disparity existed. The use of phenotypic anchoring in toxicity studies is clearly not novel (Moggs et al., 2004; Naciff et al., 2007; Powell et al., 2006); however, unlike previous publications, we have applied this approach to standard repeat-dose studies using a range of dose levels with the intention of establishing NOAELs based on phenotypic and molecular changes. Two independent 28-day toxicity studies, spaced 1 year apart, investigated the effects of FM at different dose levels (0.04–150 mg/kg/day). For one of these studies, proteomic and histopathological investigation of the testes and measurement of plasma testosterone levels had previously been conducted in our laboratory following treatment of rats with FM for 28 days at doses of 6, 30, and 150 mg/kg/day (Friry-Santini et al., 2007). For the present investigations, testicular samples from these same animals were evaluated for gene expression changes using quantitative PCR (qPCR). An additional 28-day study was subsequently conducted in which rats were treated with lower doses of FM (0.04, 0.2, 1, and 6 mg/kg/day). At necropsy, plasma testosterone levels were measured and the testes were collected for histopathological assessment as well as for the evaluation of gene expression changes using qPCR analyses. Our results showed that in a standard repeat-dose toxicity study investigating FM, the NOAELs based on testicular histopathology and plasma testosterone measurements are similar to the NOAEL based on transcript changes in the testis. MATERIALS AND METHODS Animals and Housing Male Sprague-Dawley rats were housed and maintained as described previously (Friry-Santini et al., 2007). Dosing and Experimental Design FM (CAS number 034K1459; Sigma, Saint Quentin Fallavier, France) suspended in 0.5% methylcellulose was administered to rats (6 weeks old at start of treatment and six per group) orally by gavage at a daily dose of 0 (control), 0.04, 0.2, 1, or 6 mg/kg body weight, for 28 consecutive days, using a dose volume of 5 ml/kg body weight. Methylcellulose in sterilized water (0.5% wt/vol) was used as the control vehicle. Clinical observations were performed daily, and body weights and physical examinations were recorded weekly. Twenty-four hours after the last dose, all animals were killed by isoflurane (Baxter, Maurepas, France) inhalation followed by exsanguination under deep anesthesia. Plasma samples were prepared from terminal blood samples from each animal and stored at −80°C until hormone analysis. At necropsy, each left testis was decapsulated and cut into three equal parts, which were then flash frozen in liquid nitrogen prior to storing at −80°C. Histopathology The right testis was fixed for 72 h in 10% neutral buffered formalin fixative. Paraffin-embedded tissue was prepared, sectioned at about 5 μm, and stained with hematoxylin (Sigma) and eosin (Merck, Fontenay-sous-Bois, France) for histological examination under light microscopy. The severity of Leydig cell hyperplasia was graded as described previously (Friry-Santini et al., 2007). Hormone Measurements Testosterone levels were determined in individual plasma samples using a specific radioimmunoassay kit (Beckman Coulter, Villepinte, France). Molecular Analyses To provide a genomic profile anchored to clear phenotypic changes (i.e., Leydig cell hyperplasia), samples of control and FM (6, 30, and 150 mg/kg/day) -treated testes from a previous study (Friry-Santini et al., 2007) were included in the manipulations. Portions of these tissues had previously been used for global proteomic profiling, and the data have been reported (Friry-Santini et al., 2007). Total RNA isolation. Total RNA was isolated from individual control and treated testicular samples of the present study and from those reported previously in Friry-Santini et al. (2007) using RNeasy Midi kits (Qiagen, Valencia, CA). Quality controls were performed based on the ribosomal RNA electrophoretic profiles using a Bioanalyser (Agilent Technologies, Santa Clara, CA). qPCR analysis. The selection of genes was based on a preliminary full-genome transcriptome analysis of testicular RNA samples of control and 150 mg/kg/day FM-treated animals (Friry-Santini et al., 2007). As the objective of this paper was to establish NOAELs for the testicular toxicity induced by FM, we focused on those biological pathways whose functions were unequivocally linked to the testicular lesion observed following 28-day treatment with 150 mg/kg/day FM. Thus, genes involved in lipid metabolism (for testosterone synthesis by Leydig cells) and cell proliferation/cell death (for Leydig cell hyperplasia) were investigated. Specifically, we selected Hmgcs1, Hsd17b7, Ebp, Dhcr7, Star, Cyp17a1, Grlac, and Fabp3 for lipid metabolism. For cell proliferation, Ccnd3, Myc, Prlr, Fdxr, and G6pdx were selected, and for cell death/apoptosis, Casp3, Casp6, Hsp60, and Fdxr were selected. Finally, a number of other genes were also evaluated that were not directly associated with the observed testicular lesion. Thus, genes involved in amino acid/protein metabolism (Pah and Hp) and genes involved in carbohydrate metabolism and glycolysis (Idh1 and Mdh1, respectively) were assessed. Ten micrograms of pooled or individual total RNA from control and treated testes from the present and the previously reported study (Friry-Santini et al., 2007) were used for reverse transcription using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). The qPCR assays were performed in duplicate using Taqman probes (Assay on demand; Applied Biosystems), 1/50 diluted first-strand complementary DNA, AmpliTaq Gold PCR Master Mix on an ABI prism 7900 HT machine (Applied Biosystems). b-actin was selected as reference gene for the quantitative calculations. Using pools of total RNA samples, genes were considered altered if the fold change was < 0.66 or > 1.5. Statistical Analyses Statistical analyses on body and organ weights and hormonal parameters were performed as previously described (Kennel et al., 2004). T-test statistical analysis was performed on qPCR analysis on individual total RNA samples using a cutoff value of p < 0.01 to consider a gene altered. RESULTS Standard Toxicological Measurements Following 28-day FM exposure, testicular histopathological analysis revealed Leydig cell hyperplasia only at doses of 6 mg/kg/day and above (Table 1), with the severity of the lesion increasing with increasing dose level. Similarly, a dose-related increase in plasma testosterone level was observed from the dose of 6 mg/kg/day and above (Table 2). In addition, the weight of the androgen-dependent tissues was decreased by FM exposure (Table 2). This effect was detected at 30 and 150 mg/kg/day for the ventral prostate and at 6 mg/kg/day and above for the epididymis and the seminal vesicles. Terminal body weight and testis weight were not affected at any dose level. TABLE 1 Incidence and Severity of Leydig Cell Hyperplasia. Data are Presented as the Number of Animals Affected in Each Group FM (mg/kg/day) Severity 0 0.04 0.20 1 6 30 150 Present study Minimal (grade 1) 0 0 0 0 6 Slight (grade 2) 0 0 0 0 0 Moderate (grade 3) 0 0 0 0 0 Marked (grade 4) 0 0 0 0 0 Total 0/6 0/6 0/6 0/6 6/6 Friry-Santini et al. (2007), study 2 Minimal (grade 1) 0 4 1 0 Slight (grade 2) 0 1 3 1 Moderate (grade 3) 0 0 2 3 Marked (grade 4) 0 0 0 2 Total 0/6 5/6 6/6 6/6 FM (mg/kg/day) Severity 0 0.04 0.20 1 6 30 150 Present study Minimal (grade 1) 0 0 0 0 6 Slight (grade 2) 0 0 0 0 0 Moderate (grade 3) 0 0 0 0 0 Marked (grade 4) 0 0 0 0 0 Total 0/6 0/6 0/6 0/6 6/6 Friry-Santini et al. (2007), study 2 Minimal (grade 1) 0 4 1 0 Slight (grade 2) 0 1 3 1 Moderate (grade 3) 0 0 2 3 Marked (grade 4) 0 0 0 2 Total 0/6 5/6 6/6 6/6 Note. Grading of severity is as described in Materials and Methods. Briefly, minimal (grade 1): increase of Leydig cells within the interstitial space; slight (grade 2): complete replacement of the interstitial space; moderate (grade 3): presence of bridging strands of Leydig cells surrounding the seminiferous tubules (one or two cell layers); marked (grade 4): presence of bridging strands of Leydig cells surrounding the seminiferous tubules (more than two cell layers). Open in new tab TABLE 1 Incidence and Severity of Leydig Cell Hyperplasia. Data are Presented as the Number of Animals Affected in Each Group FM (mg/kg/day) Severity 0 0.04 0.20 1 6 30 150 Present study Minimal (grade 1) 0 0 0 0 6 Slight (grade 2) 0 0 0 0 0 Moderate (grade 3) 0 0 0 0 0 Marked (grade 4) 0 0 0 0 0 Total 0/6 0/6 0/6 0/6 6/6 Friry-Santini et al. (2007), study 2 Minimal (grade 1) 0 4 1 0 Slight (grade 2) 0 1 3 1 Moderate (grade 3) 0 0 2 3 Marked (grade 4) 0 0 0 2 Total 0/6 5/6 6/6 6/6 FM (mg/kg/day) Severity 0 0.04 0.20 1 6 30 150 Present study Minimal (grade 1) 0 0 0 0 6 Slight (grade 2) 0 0 0 0 0 Moderate (grade 3) 0 0 0 0 0 Marked (grade 4) 0 0 0 0 0 Total 0/6 0/6 0/6 0/6 6/6 Friry-Santini et al. (2007), study 2 Minimal (grade 1) 0 4 1 0 Slight (grade 2) 0 1 3 1 Moderate (grade 3) 0 0 2 3 Marked (grade 4) 0 0 0 2 Total 0/6 5/6 6/6 6/6 Note. Grading of severity is as described in Materials and Methods. Briefly, minimal (grade 1): increase of Leydig cells within the interstitial space; slight (grade 2): complete replacement of the interstitial space; moderate (grade 3): presence of bridging strands of Leydig cells surrounding the seminiferous tubules (one or two cell layers); marked (grade 4): presence of bridging strands of Leydig cells surrounding the seminiferous tubules (more than two cell layers). Open in new tab TABLE 2 Effects of FM on Mean Terminal Body Weight, Sex Organs, Accessory Tissue Weights, and Plasma Testosterone Levels FM (mg/kg/day) Study 0 0.04 0.20 1 6 30 150 Terminal body weight (g) 1 374.3 ± 19.8 373.5 ± 28.8 (0) 371.9 ± 35.4 (−1) 359.2 ± 23.4 (−4) 358.2 ± 16.0 (−4) 2 363.0 ± 29.0 354.0 ± 20.0 (−2) 357.0 ± 25.0 (−2) 328.0 ± 25.0 (−10) Testis (g) 1 1.63 ± 0.11 1.72 ± 0.07 (+5) 1.63 ± 0.12 (0) 1.66 ± 0.16 (+2) 1.75 ± 0.16 (+7) 2 1.73 ± 0.11 1.72 ± 0.07 (−1) 1.82 ± 0.09 (+5) 1.64 ± 0.45 (−5) Epididymis (g) 1 0.46 ± 0.07 0.43 ± 0.02 (−7) 0.41 ± 0.04 (−10) 0.39 ± 0.08 (−16) 0.36* ± 0.03 (−21) 2 0.57 ± 0.02 0.48* ± 0.07 (−15) 0.43** ± 0.03 (−25) 0.25** ± 0.08 (−56) Ventral prostate (g) 1 0.38 ± 0.03 0.44 ± 0.08 (+17) 0.45 ± 0.09 (+19) 0.39 ± 0.13 (+3) 0.30 ± 0.09 (−21) 2 0.60 ± 0.17 0.44 ± 0.04 (−26) 0.24* ± 0.04 (−61) 0.12** ± 0.07 (−80) Seminal vesicles (g) 1 1.22 ± 0.18 1.06 ± 0.18 (−13) 1.01 ± 0.08 (−17) 0.97 ± 0.35 (−21) 0.63* ± 0.13 (−49) 2 1.35 ± 0.36 0.90 ± 0.12 (−33) 0.58* ± 0.20 (−57) 0.13** ± 0.03 (−90) Plasma testosterone levels (ng/ml) 1 2.40 ± 1.63 2.74 ± 2.45 (+14) 2.78 ± 1.66 (+16) 2.74 ± 1.37 (+14) 8.62** ± 2.23 (+ 259) 2 1.44 ± 1.10 6.02** ± 3.11 (+ 311) 12.42** ± 6.54 (+ 763) 17.69** ± 6.45 (+ 1128) FM (mg/kg/day) Study 0 0.04 0.20 1 6 30 150 Terminal body weight (g) 1 374.3 ± 19.8 373.5 ± 28.8 (0) 371.9 ± 35.4 (−1) 359.2 ± 23.4 (−4) 358.2 ± 16.0 (−4) 2 363.0 ± 29.0 354.0 ± 20.0 (−2) 357.0 ± 25.0 (−2) 328.0 ± 25.0 (−10) Testis (g) 1 1.63 ± 0.11 1.72 ± 0.07 (+5) 1.63 ± 0.12 (0) 1.66 ± 0.16 (+2) 1.75 ± 0.16 (+7) 2 1.73 ± 0.11 1.72 ± 0.07 (−1) 1.82 ± 0.09 (+5) 1.64 ± 0.45 (−5) Epididymis (g) 1 0.46 ± 0.07 0.43 ± 0.02 (−7) 0.41 ± 0.04 (−10) 0.39 ± 0.08 (−16) 0.36* ± 0.03 (−21) 2 0.57 ± 0.02 0.48* ± 0.07 (−15) 0.43** ± 0.03 (−25) 0.25** ± 0.08 (−56) Ventral prostate (g) 1 0.38 ± 0.03 0.44 ± 0.08 (+17) 0.45 ± 0.09 (+19) 0.39 ± 0.13 (+3) 0.30 ± 0.09 (−21) 2 0.60 ± 0.17 0.44 ± 0.04 (−26) 0.24* ± 0.04 (−61) 0.12** ± 0.07 (−80) Seminal vesicles (g) 1 1.22 ± 0.18 1.06 ± 0.18 (−13) 1.01 ± 0.08 (−17) 0.97 ± 0.35 (−21) 0.63* ± 0.13 (−49) 2 1.35 ± 0.36 0.90 ± 0.12 (−33) 0.58* ± 0.20 (−57) 0.13** ± 0.03 (−90) Plasma testosterone levels (ng/ml) 1 2.40 ± 1.63 2.74 ± 2.45 (+14) 2.78 ± 1.66 (+16) 2.74 ± 1.37 (+14) 8.62** ± 2.23 (+ 259) 2 1.44 ± 1.10 6.02** ± 3.11 (+ 311) 12.42** ± 6.54 (+ 763) 17.69** ± 6.45 (+ 1128) Note. Data are presented as the mean ± SD. Values in brackets refer to the percentage weight difference compared to control. Study 1 refers to the present study and study 2 refers to the study by Friry-Santini et al. (2007). *p < 0.05. **p < 0.01. Open in new tab TABLE 2 Effects of FM on Mean Terminal Body Weight, Sex Organs, Accessory Tissue Weights, and Plasma Testosterone Levels FM (mg/kg/day) Study 0 0.04 0.20 1 6 30 150 Terminal body weight (g) 1 374.3 ± 19.8 373.5 ± 28.8 (0) 371.9 ± 35.4 (−1) 359.2 ± 23.4 (−4) 358.2 ± 16.0 (−4) 2 363.0 ± 29.0 354.0 ± 20.0 (−2) 357.0 ± 25.0 (−2) 328.0 ± 25.0 (−10) Testis (g) 1 1.63 ± 0.11 1.72 ± 0.07 (+5) 1.63 ± 0.12 (0) 1.66 ± 0.16 (+2) 1.75 ± 0.16 (+7) 2 1.73 ± 0.11 1.72 ± 0.07 (−1) 1.82 ± 0.09 (+5) 1.64 ± 0.45 (−5) Epididymis (g) 1 0.46 ± 0.07 0.43 ± 0.02 (−7) 0.41 ± 0.04 (−10) 0.39 ± 0.08 (−16) 0.36* ± 0.03 (−21) 2 0.57 ± 0.02 0.48* ± 0.07 (−15) 0.43** ± 0.03 (−25) 0.25** ± 0.08 (−56) Ventral prostate (g) 1 0.38 ± 0.03 0.44 ± 0.08 (+17) 0.45 ± 0.09 (+19) 0.39 ± 0.13 (+3) 0.30 ± 0.09 (−21) 2 0.60 ± 0.17 0.44 ± 0.04 (−26) 0.24* ± 0.04 (−61) 0.12** ± 0.07 (−80) Seminal vesicles (g) 1 1.22 ± 0.18 1.06 ± 0.18 (−13) 1.01 ± 0.08 (−17) 0.97 ± 0.35 (−21) 0.63* ± 0.13 (−49) 2 1.35 ± 0.36 0.90 ± 0.12 (−33) 0.58* ± 0.20 (−57) 0.13** ± 0.03 (−90) Plasma testosterone levels (ng/ml) 1 2.40 ± 1.63 2.74 ± 2.45 (+14) 2.78 ± 1.66 (+16) 2.74 ± 1.37 (+14) 8.62** ± 2.23 (+ 259) 2 1.44 ± 1.10 6.02** ± 3.11 (+ 311) 12.42** ± 6.54 (+ 763) 17.69** ± 6.45 (+ 1128) FM (mg/kg/day) Study 0 0.04 0.20 1 6 30 150 Terminal body weight (g) 1 374.3 ± 19.8 373.5 ± 28.8 (0) 371.9 ± 35.4 (−1) 359.2 ± 23.4 (−4) 358.2 ± 16.0 (−4) 2 363.0 ± 29.0 354.0 ± 20.0 (−2) 357.0 ± 25.0 (−2) 328.0 ± 25.0 (−10) Testis (g) 1 1.63 ± 0.11 1.72 ± 0.07 (+5) 1.63 ± 0.12 (0) 1.66 ± 0.16 (+2) 1.75 ± 0.16 (+7) 2 1.73 ± 0.11 1.72 ± 0.07 (−1) 1.82 ± 0.09 (+5) 1.64 ± 0.45 (−5) Epididymis (g) 1 0.46 ± 0.07 0.43 ± 0.02 (−7) 0.41 ± 0.04 (−10) 0.39 ± 0.08 (−16) 0.36* ± 0.03 (−21) 2 0.57 ± 0.02 0.48* ± 0.07 (−15) 0.43** ± 0.03 (−25) 0.25** ± 0.08 (−56) Ventral prostate (g) 1 0.38 ± 0.03 0.44 ± 0.08 (+17) 0.45 ± 0.09 (+19) 0.39 ± 0.13 (+3) 0.30 ± 0.09 (−21) 2 0.60 ± 0.17 0.44 ± 0.04 (−26) 0.24* ± 0.04 (−61) 0.12** ± 0.07 (−80) Seminal vesicles (g) 1 1.22 ± 0.18 1.06 ± 0.18 (−13) 1.01 ± 0.08 (−17) 0.97 ± 0.35 (−21) 0.63* ± 0.13 (−49) 2 1.35 ± 0.36 0.90 ± 0.12 (−33) 0.58* ± 0.20 (−57) 0.13** ± 0.03 (−90) Plasma testosterone levels (ng/ml) 1 2.40 ± 1.63 2.74 ± 2.45 (+14) 2.78 ± 1.66 (+16) 2.74 ± 1.37 (+14) 8.62** ± 2.23 (+ 259) 2 1.44 ± 1.10 6.02** ± 3.11 (+ 311) 12.42** ± 6.54 (+ 763) 17.69** ± 6.45 (+ 1128) Note. Data are presented as the mean ± SD. Values in brackets refer to the percentage weight difference compared to control. Study 1 refers to the present study and study 2 refers to the study by Friry-Santini et al. (2007). *p < 0.05. **p < 0.01. Open in new tab Molecular Measurements Testicular qPCR Analysis At 1 mg/kg/day and below, there were no changes in gene transcript levels recorded using pooled RNA samples (Fig. 1) in either of the classes of gene studied (lipid metabolism and cell proliferation/cell death) as well as in those genes not directly associated with the testicular lesion. This was also true for those genes, which were investigated on an individual animal basis (Fig. 2). These molecular data supported the phenotypic data as no lesions and no changes in plasma testosterone levels were detected at 1 mg/kg/day and below. FIG. 1. Open in new tabDownload slide The qPCR data using pooled RNA samples for genes associated with (a) lipid metabolism, (b) cell proliferation and cell death, and (c) biological functions not related to the observed testicular lesion. FIG. 1. Open in new tabDownload slide The qPCR data using pooled RNA samples for genes associated with (a) lipid metabolism, (b) cell proliferation and cell death, and (c) biological functions not related to the observed testicular lesion. FIG. 2. Open in new tabDownload slide The qPCR data using individual RNA samples for genes associated with lipid metabolism (Cyp17a1, Star, and Hsd17b7) and with cell proliferation and cell death (Prlr, Ccnd3, and Myc). FIG. 2. Open in new tabDownload slide The qPCR data using individual RNA samples for genes associated with lipid metabolism (Cyp17a1, Star, and Hsd17b7) and with cell proliferation and cell death (Prlr, Ccnd3, and Myc). At 6 mg/kg/day, alterations in some of the genes associated with lipid metabolism (Lss, Dhcr7, Star, and Cyp17a1) and cell proliferation/cell death (Prdx3, Fdxr, G6pdx, and Prlr) were recorded using pooled RNA samples (Fig. 1). The individual animal data generated for Star, Cyp17a1, and Prdx3 gene expression were also changed when investigated on an individual animal basis. Interestingly, Hp, which is not linked to the testicular lesion, was also upregulated at 6 mg/kg/day. All other genes associated with lipid metabolism and cell proliferation/cell death were deregulated from 30 mg/kg/day (Fig. 1), with the exception of Hmgcs1 and Ebp1, which were found altered only at the high dose of 150 mg/kg/day FM. All genes not associated with the testicular lesion were altered from 30 mg/kg/day with the exception of Idh1, which was upregulated at 150 mg/kg/day. Pah was the only gene in these investigations that was downregulated with the alteration occurring at 30 mg/kg/day and above. The dose at which transcript changes were first observed (i.e., 6 mg/kg/day), using samples of pooled RNA, was the same as that at which histopathological and hormonal changes were first observed. In addition, the changes recorded using the pooled RNA samples were supported by the changes recorded for the individual animals at 6 mg/kg/day and below. For example, deregulation of Cyp17a1, Star, and Prlr genes was observed in both the pooled and the individual samples starting at the dose of 6 mg/kg/day FM. In addition, Hsd17b7 and Myc, which were deregulated in pooled samples at 30 and 150 mg/kg/day FM, were not altered up to 6 mg/kg/day FM using the individual samples. Based on both the pooled and the individual data, statistically significant deregulation (p < 0.05) of transcripts was only obtained at a dose of 6 mg/kg/day FM and above. DISCUSSION We have shown that in a standard repeat-dose toxicity study investigating the effects of FM on the testes, the dose level at which treatment related changes were observed was the same for histopathological examination, hormone level measurements, and transcript changes. As a result, we established a NOAEL of 1 mg/kg/day for FM for each parameter measured, which, for the standard measurements, is in agreement with previously published data (Andrews et al., 2001; Friry-Santini et al., 2007; Kunimatsu et al., 2004; Tinwell et al., 2007; Toyoda et al., 2000). For the qPCR analysis, we selected a number of genes known to be involved in the metabolic pathways associated with the testicular lesion induced by FM based on our previous experiences with the compound (Friry-Santini et al., 2007). In addition, a number of genes not directly associated with the testicular lesion were also investigated. An interesting aspect of these data concerns the similarity of the qPCR data, which were generated for the same dose level of 6 mg/kg/day in two independent studies conducted approximately 12 months apart (Fig. 1). These data demonstrate an excellent reproducibility for the transcript changes between the two studies. Our studies indicated that histopathological and hormonal changes were recorded at a dose of 6 mg/kg/day and that increases in the severity of the testicular lesion as well as increases in plasma testosterone concentration were recorded with increasing dose. Similar observations were recorded for the gene expression data inasmuch as gene deregulation was first recorded at a dose of 6 mg/kg/day FM, and the number of deregulated genes in a given metabolic pathway (lipid metabolism and cell proliferation/cell death) increased with increasing dose level. For example, for the genes involved in the lipid metabolism, only Star and Cyp17a1 were significantly deregulated at 6 mg/kg/day, despite Leydig cell hyperplasia and significant increases in plasma testosterone being recorded. Deregulation of Hsd17b7, Lss, Grlacs, and Fabp3 started at 30 mg/kg/day FM, whereas Hmgcs1 was altered only at 150 mg/kg/day FM. Similarly, deregulation of those genes with no direct association with the testicular lesion began at 6 mg/kg/day. One possible limitation of our study is the small number of animals per group (n = 6). Indeed, it cannot be excluded that using a larger group size, changes in all three parameters investigated would be detected at lower dose levels. However, as the objective of this study was to establish, under standard conditions of test, NOAELs for FM based on histopathological, hormonal, and transcriptional changes, the group sizes were in accordance with those used in standard toxicity studies. Another possible limitation lies in the relatively few genes chosen for the molecular investigation using qPCR analysis. The genes were selected according to the available literature concerning the molecular effect of FM on the testis (Ohsako et al., 2003) as well as the effect of FM on testosterone biosynthesis. A complete full-genome analysis of all the testicular samples described herein would clearly allow the identification of additional classes of genes modulated at specific dose levels. In addition, the lesion studied in this toxicity study occurs in a specific cell type in the testis, i.e., the Leydig cell, which is responsible for testosterone biosynthesis. The gene changes assessed in our particular studies were investigated using the whole testis, of which the Leydig cells comprise only a minor fraction. Obviously, techniques such as cell fractionation (Boussouar et al., 2003; Goddard et al., 2003; Mauduit et al., 2001) and laser microdissection (Plummer et al., 2007) may prove useful to identify those genes uniquely modulated in the Leydig cells. To our knowledge, this is the first report that has determined NOAELs for histopathological, hormonal, and transcriptional changes in an endocrine tissue using a standard repeat-dose toxicity study. A similar approach has recently been reported for the effects of formaldehyde in the rat nasal tissue. Similar to our data, the authors observed statistically significant gene alterations only at those dose levels for which cell proliferation or a nasal lesion was detected (Andersen et al., 2008). In conclusion, our results demonstrate that, in a standard repeat-dose toxicity study, the dose at which toxicity was detected was similar between histopathological examination, hormone level measurements, and transcript changes. Following on from these observations, future research should be directed towards a more detailed molecular analysis of the testes for all the dose levels of FM used in our current studies to confirm the observations reported herein. In addition, this type of investigation should be expanded to other target organs and lesions to determine whether the NOAELs for FM are similar between histopathology and gene changes in other tissues as appears to be the case for the testis. For example, atrophy of the prostate is routinely observed following treatment of rats with FM (Friry-Santini et al., 2007; Tinwell et al., 2007) and would, therefore, be worthy of investigation. Finally, investigation of compounds with different mechanisms of action of toxicity and using a similar approach to that described herein is essential to determine whether our observations are unique to FM-induced testicular toxicity or more prevalent. The authors would like to thank Marie Pierre Come and Hélène Lormeau for expert assistance in qPCR analysis and Valérie Pozzoli-Cipolla for technical expertise in testosterone level measurements. C.F.-S. was recipient of a fellowship co-sponsored by the Association Nationale de la Recherche Technique and Bayer CropScience. References Andersen ME , Clewell HJ III , Bermudez E , Willson GA , Thomas RS . Genomic signatures and dose-dependent transitions in nasal epithelial responses to inhaled formaldehyde in the rat , Toxicol. Sci. , 2008 , vol. 105 (pg. 368 - 383 ) Google Scholar Crossref Search ADS PubMed WorldCat Andrews P , Freyberger A , Hartmann E , Eiben R , Loof I , Schmidt U , Temerowski M , Becka M . 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 , Arch. Toxicol. , 2001 , vol. 75 (pg. 65 - 73 ) Google Scholar Crossref Search ADS PubMed WorldCat Boussouar F , Mauduit C , Tabone E , Pellerin L , Magistretti PJ , Benahmed M . Developmental and hormonal regulation of the monocarboxylate transporter 2 (MCT2) expression in the mouse germ cells , Biol. Reprod. , 2003 , vol. 69 (pg. 1069 - 1078 ) Google Scholar Crossref Search ADS PubMed WorldCat Cunningham ML , Bogdanffy MS , Zacharewski TR , Hines RN . Workshop overview: Use of genomic data in risk assessment , Toxicol. Sci. , 2003 , vol. 73 (pg. 209 - 215 ) Google Scholar Crossref Search ADS PubMed WorldCat Freeman K . Toxicogenomics data: The road to acceptance , Environ. Health Perspect. , 2004 , vol. 112 (pg. A678 - A685 ) Google Scholar Crossref Search ADS PubMed WorldCat Friry-Santini C , Rouquié D , Kennel P , Tinwell H , Benahmed M , Bars R . Correlation between protein accumulation profiles and conventional toxicological findings using a model antiandrogenic compound, flutamide , Toxicol. Sci. , 2007 , vol. 97 (pg. 81 - 93 ) Google Scholar Crossref Search ADS PubMed WorldCat Goddard I , Florin A , Mauduit C , Tabone E , Contard P , Bars R , Chuzel F , Benahmed M . Alteration of lactate production and transport in the adult rat testis exposed in utero to flutamide , Mol. Cell. Endocrinol. , 2003 , vol. 206 (pg. 137 - 146 ) Google Scholar Crossref Search ADS PubMed WorldCat Kennel PF , Pallen CT , Bars RG . Evaluation of the rodent Hershberger assay using three reference endocrine disrupters (androgen and antiandrogens) , Reprod. Toxicol. , 2004 , vol. 18 (pg. 63 - 73 ) Google Scholar Crossref Search ADS PubMed WorldCat Kunimatsu T , Yamada T , Miyata K , Yabushita S , Seki T , Okuno Y , Matsuo M . Evaluation for reliability and feasibility of the draft protocol for the enhanced rat 28-day subacute study (OECD Guideline 407) using androgen antagonist flutamide , Toxicology , 2004 , vol. 200 (pg. 77 - 89 ) Google Scholar Crossref Search ADS PubMed WorldCat Mauduit C , Goddard I , Besset V , Tabone E , Rey C , Gasnier F , Dacheux F , Benahmed M . Leukemia inhibitory factor antagonizes gonadotropin induced-testosterone synthesis in cultured porcine leydig cells: Sites of action , Endocrinology , 2001 , vol. 142 (pg. 2509 - 2520 ) OpenURL Placeholder Text WorldCat Moggs JG , Tinwell H , Spurway T , Chang HS , Pate I , Lim FL , Moore DJ , Soames A , Stuckey R , Currie R , et al. Phenotypic anchoring of gene expression changes during estrogen-induced uterine growth , Environ. Health Perspect. , 2004 , vol. 112 (pg. 1589 - 1606 ) Google Scholar Crossref Search ADS PubMed WorldCat Naciff JM , Overmann GJ , Torontali SM , Carr GJ , Khambatta ZS , Tiesman JP , Richardson BD , Daston GP . Uterine temporal response to acute exposure to 17alpha-ethinyl estradiol in the immature rat , Toxicol. Sci. , 2007 , vol. 97 (pg. 467 - 490 ) Google Scholar Crossref Search ADS PubMed WorldCat Ohsako S , Kubota K , Kurosawa S , Takeda K , Qing W , Ishimura R , Tohyama C . Alterations of gene expression in adult male rat testis and pituitary shortly after subacute administration of the antiandrogen flutamide , J. Reprod. Dev. , 2003 , vol. 49 (pg. 275 - 290 ) Google Scholar Crossref Search ADS PubMed WorldCat Plummer S , Sharpe RM , Hallmark N , Mahood IK , Elcombe C . Time-dependent and compartment-specific effects of in utero exposure to Di(n-butyl) phthalate on gene/protein expression in the fetal rat testis as revealed by transcription profiling and laser capture microdissection , Toxicol. Sci. , 2007 , vol. 97 (pg. 520 - 532 ) Google Scholar Crossref Search ADS PubMed WorldCat Powell CL , Kosyk O , Ross PK , Schoonhoven R , Boysen G , Swenberg JA , Heinloth AN , Boorman GA , Cunningham ML , Paules RS , et al. Phenotypic anchoring of acetaminophen-induced oxidative stress with gene expression profiles in rat liver , Toxicol. Sci. , 2006 , vol. 93 (pg. 213 - 222 ) Google Scholar Crossref Search ADS PubMed WorldCat Tinwell H , Friry-Santini C , Rouquie D , Belluco S , Elies L , Pallen C , Bars R . Evaluation of the antiandrogenic effects of flutamide, DDE, and linuron in the weanling rat assay using organ weight, histopathological, and proteomic approaches , Toxicol. Sci. , 2007 , vol. 100 (pg. 54 - 65 ) Google Scholar Crossref Search ADS PubMed WorldCat Toyoda K , Shibutani M , Tamura T , Koujitani T , Uneyama C , Hirose M . 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 , Arch. Toxicol. , 2000 , vol. 74 (pg. 127 - 132 ) Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
TI - Standard and Molecular NOAELs for Rat Testicular Toxicity Induced by Flutamide
JF - Toxicological Sciences
DO - 10.1093/toxsci/kfp056
DA - 2009-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/standard-and-molecular-noaels-for-rat-testicular-toxicity-induced-by-r8GKNm7j0T
SP - 59
EP - 65
VL - 109
IS - 1
DP - DeepDyve
ER -