Toxicogenomic investigation on rat testicular toxicity elicited by 1,3-dinitrobenzene

Toxicology 290 (2011) 169–177

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Toxicogenomic investigation on rat testicular toxicity elicited by 1,3-dinitrobenzene Takuya Matsuyama ∗ , Noriyo Niino, Naoki Kiyosawa, Kiyonori Kai, Munehiro Teranishi, Atsushi Sanbuissho Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan

a r t i c l e

i n f o

Article history: Received 14 June 2011 Received in revised form 31 August 2011 Accepted 4 September 2011 Available online 22 September 2011 Keywords: Testicular toxicity 1,3-Dinitrobenzene Microarray Biomarker Toxicogenomics

a b s t r a c t Rats were treated with a single oral dose of 10, 25 and 50 mg/kg of 1,3-dinitrobenzene (DNB), and the testis was subjected to a GeneChip microarray analysis. A total of 186 and 304 gene probe sets were up- and down-regulated, respectively, by the DNB treatment, where spermatocyte death and Sertoli cell vacuolation in testis and increased debris of spermatogenic cell in epididymis were noted. The expression profile for four sets of genes were investigated, whose expressions are reported to localize in specific cell types in the seminiferous epithelium, namely Sertoli cells, spermatogonia plus early spermtocytes, pachytene spermatocytes and round spermatids. The data demonstrated that pachytene spermatocyte-specific genes elicited explicit down-regulation in parallel with the progression of spermatocyte death, while other gene sets did not show characteristic expression changes. In addition, Gene Ontology analysis indicated that genes associated with cell adhesion-related genes were significantly enriched in the up-regulated genes following DNB treatment. Cell adhesion-related genes, namely Cdh2, Ctnna1, Vcl, Zyx, Itgb1, Testin, Lamc3, Pvrl2 and Gsn, showed an increase in microarray and the up-regulation of Cdh2 and Testin were confirmed by real time RT-PCR. The gene expression changes of pachytene spermatocyte-specific genes and cell adhesion-related genes were thought to reflect a decrease in the number of spermatocytes and dysfunction of Sertoli–germ cells adhesion junction, and therefore these genes would be potential genomic biomarkers for assessing DNB-type testicular toxicity. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Testicular toxicity is one of the major concerns in preclinical toxicity studies in the drug development process. The testis contains a variety of cell types including different phases of spermatogenic cells (spermatogonia, spermatocytes, spermatids and spermatozoa), Sertoli cells and Leydig cells, and these cells are affected by several hormones. Due to the structural and functional complexity of testis, characterization of testicular toxicity, such as identifying target cells or a mechanism of action, is relatively difficult. Dinitrobenzene is a nitroaromatic compound used in the synthesis of dyes intermediate, in the plastics industry and as an explosive, and 1,3-dinitrobenzene (DNB) is an isomer shown as a testicular toxicant (Blackburn et al., 1988). Subchronic exposures of DNB cause testicular atrophy and histopathological changes such as a decrease in the number of sperm-spermatocytes, degeneration/necrosis, giant cell formation and vacuolation (Cody et al., 1981; Irimura et al., 2000). Early reactions from DNB in testis

∗ Corresponding author. Tel.: +81 3 3680 0151; fax: +81 3 5696 8335. E-mail address: [email protected] (T. Matsuyama). 0300-483X/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2011.09.001

include the injury to Sertoli cells and induction of germ cell apoptosis (Blackburn et al., 1988; Hess et al., 1988; Strandgaard and Miller, 1998). In addition, germ cell sloughing is reported to be a characteristic finding in the DNB-induced testicular toxicity, which was shown in some studies in in vivo or in vitro (Foster et al., 1987; Hess et al., 1988). Sertoli cells have been suggested as an initial target of DNB and the germ cell damage is thought to be a secondary event, based on the finding that the vacuolation of Sertoli cells was observed by an ultrastructural examination before the morphological changes in the germ cells (Blackburn et al., 1988). As for the possible mechanism underlying DNB-induced testicular toxicity, it is thought that nitroreduction of DNB within the seminiferous tubules might be causing testicular toxicity by compromising mitochondrial antioxidant capacity (Jacobson and Miller, 1998; Reeve et al., 2002). At the transcriptional level, gene expression analysis of apoptosis-related gene (i.e., Bax, Bcl-2, BclxL, and Bcl-xs) by RT-PCR methods indicated that the germ cell apoptosis in the testis of DNB-treated rats is induced via the mitochondrial pathway (Muguruma et al., 2005). However, a detailed molecular mechanism to explain the above-mentioned characteristic phenotypes of DNB-induced toxicity has not been well understood, and thus it is expected that a comprehensive gene

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expression analysis using microarray technique could enable us to better characterize the DNB-induced testicular toxicity at a molecular level. In both the preclinical and the clinical toxicity evaluation, various types of biomarkers have been utilized which are either directly or indirectly associated with drug-induced toxicity endpoints. Utilization of such traditional biomarkers in conjunction with the histopathological examination gives us a clue to the mechanism of toxicity. However, information for detailed molecular dynamics is relatively limited and accordingly the uncertainty of drug-induced toxicity could sometimes give a confounding impact on further drug development stages. In the last decade, the technology of microarray, by which genome-wide gene expression levels can be measured simultaneously, has been utilized in toxicology studies, and is referred to as toxicogenomics. Advances in toxicogenomics, offers opportunities for the toxicologist to investigate detailed molecular mechanisms of toxicity and to identify novel candidate biomarkers. For example, a number of gene sets whose expression changes are closely associated with hepatotoxicity have been reported (Kiyosawa et al., 2009a), and such toxicogenomic biomarkers are now widely utilized in the pharmaceutical companies for toxicity profiling and compound selection. In the case of the preclinical evaluation of testicular toxicity, traditional biomarkers include blood hormone levels, examination of sperm, the weight of reproductive organs and histopathology evaluation, however, toxicogenomic biomarkers on testicular toxicity is considerably limited compared with those for hepatotoxicity. Recently, Johnston et al. (2008) presented information on genes whose expressions are high in the specific cell types of seminiferous cells. This report provided us with information to develop novel toxicogenomic biomarkers. In the present study, we investigated whether these genes could be useful biomarkers to evaluate which types of seminiferous cells that are damaged by the treatment of chemicals which induce testicular toxicity. Such toxicogenomic biomarkers could possess better sensitivity, considering that the gene expression profile is affected by perturbation of the cellular function in precedent with histological changes. For this purpose, we conducted a microarray analysis on rat testis treated with DNB to evaluate the usefulness of the testicular gene sets as a cell type-specific and highly sensitive biomarker. In addition, we explored potential new biomarker genes to evaluate DNB-type testicular toxicity from the differentially regulated genes. 2. Materials and methods 2.1. Animals and chemical treatment Nine-week-old male F344/DuCrlCrlj rats were purchased from Charles River Japan, Inc. (Kanagawa, Japan). The animals were housed under controlled conditions (24 ± 2 ◦ C temperature, 40–70% relative humidity, and a 12:12 light–dark cycle) and fed standard laboratory diet and water ad libitum throughout the treatment period. Animals were randomly divided into eight groups of four animals each, and treated orally with 10, 25 and 50 mg/kg of DNB (Sigma–Aldrich, St. Louis, MO, USA) or vehicle (PEG 600, Wako Pure Chemical Industries, Ltd., Osaka, Japan). Animals were sacrificed 4 or 24 h after dosing under isoflurane anesthesia. The experimental protocol was approved by the Ethics Review Committee for Animal Experimental of Daiichi Sankyo Co., Ltd. 2.2. Histopathological examination The left testis was fixed in formalin–sucrose–acetic acid fluid and the left epididymis was fixed in 10% (v/v) neutral buffered formalin for histopathological examination, respectively. The fixed testes were embedded in paraffin according to standard procedures. Histopathological specimens were prepared and stained with hematoxylin and eosin. The stages of seminiferous tubule were classified as stages I–VI, VII–VIII, IX–XI and XII–XIV and morphological changes were graded as minimal, mild, moderate and marked. Histological findings in the testis were classified and scored as follows: minimal degree (score 1), distribution up to 20% of the stage of seminiferous tubule; mild degree (score 2), distribution up to 40% of the stage of seminiferous tubule; moderate degree (score 3), distribution up to 60% of the stage of seminiferous tubule; marked degree (score 4), almost all

seminiferous tubule in the stage. Any marked changes were not observed in the present study. Histological findings in the epididymis scored as follows: minimal degree (score 1), sparse distribution of the cell debris in the duct; mild degree (score 2), cell debris sporadically found in the duct. The total scores were summed and divided by the number of animals for each group to obtain the group mean score. 2.3. RNA extraction The right testis was immediately frozen in liquid nitrogen, and stored at −80 ◦ C until use for gene expression analysis. Testis samples were homogenized with the TRIzol® Reagent (Life Technologies, Carlsbad, CA, USA), and the total RNA was isolated according to the manufacturer’s instructions. 2.4. Microarray analysis Microarray analysis was performed using GeneChip® IVT Express kit (Affymetrix Inc., Santa Clara, CA, USA) for cDNA synthesis, purification and synthesis of biotin-labeled cDNA according to the manufacturer’s instructions. Every biotinlabeled cRNA target sample (approximately 10 ␮g) was individually hybridized to a GeneChip® Rat Genome 230 2.0 Array (Affymetrix, Inc.) at 45 ◦ C for 16 h followed by washing and staining by streptavidin–phycoerythrin using Fluidics Station 400 (Affymetrix, Inc.). The scanned image was analyzed with a MAS5 algorithm using GCOS software (Affymetrix, Inc.). All the MAS5-analyzed data were scaled by global normalization. The DNB-induced up- and down-regulated genes were selected by the following criteria: (1) the detection call of each probe was “present” in >3/4 replicates, (2) the mean signal intensity values were at least 1.5-fold higher/0.67-fold lower than concurrent control value, and (3) statistical significance from concurrent control with P-value <0.05 (Student’s t-test). 2.5. Cell type-specific gene set Four cell type-specific gene sets (Sertoli cells (SC), spermatogonia plus early spermtocytes (SG), pachytene spermatocytes (PS) and round spermatids (RS)) were determined by modifying the reported gene set information (Johnston et al., 2008). These four gene sets consisted of those whose expression levels were greater by at least 4-fold in the particular cell type compared with those in other three cell types. The number of probe sets for SC-specific, SG-specific, PS-specific and RS-specific gene set are 56, 2, 41 and 269, respectively (Supplemental Tables 1–4). 2.6. Scoring the expression changes of the cell type specific gene sets Scoring the expression change of the cell type specific gene sets was performed by calculating the D-score. A detailed D-score calculation procedure has been described in a previous paper (Kiyosawa et al., 2009b). Briefly, the signal log ratio (SLR, base 2) was calculated by dividing the mean signal value of the chemicaltreated group by that of the corresponding vehicle-treated group, and the presence ratio (PR) was determined by dividing the number of the Presence Call given for both DNB- and vehicle-treated by the total number of DNB-treated and vehicletreated animals. Assuming that a gene set X consists of i probe sets (x1 , x2 , x3 , . . ., xi−1 , xi ), the calculated SLRs were given as (SLR1 , SLR2 , SLR3 , . . ., SLRi−1 , SLRi ) and PRs were given as (PR1 , PR2 , PR3 , . . ., PRi−1 , PRi ). Let the sum of (SLR1 × PR1 , SLR2 × PR2 , SLR3 × PR3 , . . ., SLRi−1 × PRi−1 , SLRi × PRi ) divided by i and the sum of ([SLR1 × PR1 ]2 , [SLR2 × PR2 ]2 , [SLR3 × PR3 ]2 , . . ., [SLRi−1 × PRi−1 ]2 , [SLRi × PRi ]2 ) divided by i, be indexes for the “overall direction of the expression change per probe set” and for the “overall magnitude of the expression change per probe set” of the Gene set X genes, respectively. The D-score is calculated by multiplying the two indexes shown above by a × 100 scaling factor. 2.7. Gene Ontology (GO) analysis GO analysis for both up- and down-regulated genes following DNB treatment was conducted using a functional annotation tool DAVID 6.7 (The Database for Annotation, Visualization and Integrated Discovery; http://david.abcc.ncifcrf.gov/). Level 3 of the biological process (GO term BP 3) was selected as an annotation category for the GO analysis. P-values were calculated using a modified Fisher Exact test in the DAVID system, and the GO terms exhibiting P < 0.05 were regarded as significantly enriched. 2.8. Real-time quantitative RT-PCR analysis Expression levels of Testin (Entrez Gene ID: 286916) and cadherin 2 (Cdh2, Entrez Gene ID: 83501) genes were measured by quantitative RT-PCR. The total RNA was treated with DNase I (Takara, Shiga, Japan) in the manufacturer’s buffer supplemented with 10 mM dithiothreitol, 0.5 mM deoxyribonucleoside triphosphates, and 40 U RNase inhibitor (Toyobo). Taqman Gene Expression Assays (for Cdh2, Assay ID Rn00580099 m1; for Testin, Assay ID: Rn00597568 m1) were used as probes and primers. The qPCR reactions were performed using qPCR Mastermix Plus (Eurogentee, Philadelphia, PA, USA). The program was 50 ◦ C for 2 min

Table 1 Histopathological findings in the rats following a single dose of DNB. 4h

24 h 10 4

25 4

Histopathological grade

NA

NA

0

Testis Stages I–VI Single cell death, spermatocyte

–

–

–

Giant cell formation

–

–

–

Vacuolation, Sertoli cell

–

–

Stages VII–VIII Single cell death, spermatocyte

50 4 1

3a

1

0 4

10 4

25 4

NA

NA

0

–

–

–

–

–

–

0

–

–

2

0

1

3

1

(0.25)

(0.25)

50 4 1

2

– 4 (1.00) 2 (0.50)

0

1

2

3

0

4

0 (1.00) –

0

0

0

4 (2.00)

0

0

4

0 (1.00) 0 (1.00)

0

2 (2.50) 0 (1.00)

2

0 0

–

–

–

–

–

–

–

–

–

–

–

–

3

1 (0.25)

0

0

4

–

–

–

–

–

–

0

0

0

0

–

–

–

–

–

–

3

4 (1.00) 1 (0.25)

0

0

4

–

–

–

–

–

–

0

3

0

0

Giant cell formation

–

–

–

–

–

–

0

Vacuolation, Sertoli cell

–

–

–

–

–

–

1 (1.75) 4 (1.00) –

–

–

–

–

–

–

1 (0.25)

0

Vacuolation, Sertoli cell Stages IX–XI Single cell death, spermatocyte Vacuolation, Sertoli cell Stages XII–XIV Single cell death, spermatocyte

Epidydimis Increased debris, spermatogenic cell

–

3

0

1 (2.75) –

0

0

3

T. Matsuyama et al. / Toxicology 290 (2011) 169–177

Numer of animals

0 4

– 0

4

0 (1.00)

0

NA: not available. – or 0: within normal limit, 1: minimal, 2: mild, 3: moderate. Parensis is showed the group mean score. a Number of animals showing the findings with each grade.

171

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Fig. 1. Light micrograph of the testis in rat 24 h after a single oral administration of vehicle or DNB at 50 mg/kg. (A) Vehicle, seminiferous tubule of stages I–III. (B) Vehicle, seminiferous tubule of stage IX. (C) Vehicle, seminiferous tubule of stages XIII–XIV. (D) DNB, seminiferous tubule of stages I–III. (E) DNB, seminiferous tubule of stage IX. (F) DNB, seminiferous tubule of stages XIII–XIV. The bar in the figure is 50 ␮m and all figures are shown at the same magnification. H&E-stained sections.

minimally in stages I–VI seminiferous tubules. However, no changes were noted in spermatogenic cells in testis and epididymis. At 24 h post-dose, single cell death of spermatocyte was noted at 25 mg/kg and it became more severe at 50 mg/kg. These findings were especially noticeable for spermatocytes at stages XII–XIV. Vacuolation of Sertoli cells were slight and observed especially at stages I–VI. In epididymis, an increased number of spermatogenic cell debris was observed at 24 h after DNB treatment at 25 and 50 mg/kg. Any histopathological changes were noted in the testis or the epididymis at 10 mg/kg. 3.2. Expression profiling of cell type-specific gene sets following DNB treatment

Fig. 2. D-score for the expression change of the four cell type-specific gene sets in testis at 4 or 24 h after a single oral administration of DNB.

and 95 ◦ C for 10 min, with amplification in 40 cycles of 95 ◦ C for 15 s and 60 ◦ C for 1 min. GAPDH (glyceraldehyde-3-phosphate dehydrogenase), of which the primers and probes were from Applied Biosystems, was used as an endogenous control.

First we tried to screen the gene set whose expression level was differentially regulated by the DNB treatment (Fig. 2), and second we tried to verify that the genes included in the gene set actually were differentially regulated (Fig. 3). The D-score of four cell type-specific gene sets are shown in Fig. 2. At 4 h after dosing, no notable changes were observed. At 24 h after dosing, PS-specific genes showed deceased D-score compared to the control group at 25 mg/kg and it became lower at 50 mg/kg, while those of SC, SG- and RS-specific gene sets showed little change. To verify the concomitant down-regulations of individual PS-specific genes, we conducted the hierarchical clustering for PS-specific genes and confirmed the concomitant down-regulations of PS-specific genes at 24 h after DNB treatment, as presented in the form of a heat map in Fig. 3. 3.3. The number of differentially regulated gene probe sets

2.9. Statistical analysis In the microarray and real-time quantitative RT-PCR analysis, the data was analyzed by an F-test to evaluate the homogeneity of variance. If the variance was homogeneous, a Student’s t-test was applied. If the variance was heterogeneous, an Aspin–Welch’s t-test was performed. In every statistical analysis, a significance level of P < 0.05 was considered statistically significant.

3. Results 3.1. Histopathology The histopathological findings were shown in Table 1 and Fig. 1. At 4 h post-dose, vacuolation of Sertoli cells was observed

The number of differentially regulated genes following DNB treatment is summarized in Fig. 4. At 4 h post dose, a total of 29 and 41 probe sets were up- and down-regulated in the 25 mg/kg DNB-treated group and a total of 149 and 71 probe sets were upand down-regulated in the 50 mg/kg DNB-treated group. At 24 h post dose, a total of 262 and 337 probe sets were up- and downregulated in the 25 mg/kg DNB-treated group and a total of 1541 and 1414 probe sets were up- and down-regulated in the 50 mg/kg DNB-treated group. In the present study, to appropriately interpret the biological significance of gene expression changes by phenotype anchoring, we focused on 24 h animal data of common to both 25 and 50 mg/kg for which histopathological changes were noted.

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Fig. 3. Gene expression profiling of pachytene spermatocytes-specific genes. The heat map represents gene expression changes, where up-regulation, no change and down-regulation are colored in red, white and blue, respectively. Data are normalized to the 4 h control mean.

For these animals, a total of 186 and 304 probe sets were identified as up- and down-regulated genes, respectively (Supplemental Tables 5 and 6).

3.4. Oxidative stress- and apoptosis-related genes in the differentially regulated genes Genes associated with oxidative stress–response (i.e., Hmox1 and Pon2), cellular protection against oxidative stress (i.e., Gstp1, Akr7a3 and Akr1b8) and apoptosis (i.e., Gadd45g, Ddit4 and Nos3) were found to be up-regulated 24 h after the DNB treatment (Table 2).

3.5. Gene Ontology analysis

Fig. 4. The number of differentially regulated gene probe sets the following DNB treatment.

To evaluate the biological characteristics, the GO analysis of the up-regulated genes and down-regulated genes at 24 h after the DNB treatment were conducted. The GO analysis showed that genes involved in several GO terms were significantly enriched in DNB induced up or down regulated genes, such as related to cell adhesion, development or cell differentiation in up-regulated genes (Table 3), locomotory behavior or cellular process in downregulated genes (Table 4).

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Table 2 Oxidative stress- and apoptosis-related genes in the differentially regulated genes after DNB treatment. Probe ID

Gene expression (/control) 4h 25 mpk

Oxidave stress-responsive 1.06 1370080 at 1.03 1375848 at Oxidave stress-protective 1.13 1388122 at 1368121 at 0.96 1370902 at 1.10 Apoptosis-related 1.25 1388792 at 1.11 1368025 at 1371166 at 1.15

Gene title

Gene symbol

24 h 50 mpk

25 mpk

50 mpk

1.11 1.02

1.61 1.67

3.20 1.78

Heme oxygenase (decycling) 1 Paraoxonase 2

Hmox1 Pon2

1.31 1.07 1.08

1.73 1.57 1.65

3.76 3.70 3.36

Glutathione-S-transferase, pi 1 Aldo-keto reductase family 7, member A3 (aflatoxin aldehyde reductase) Aldo-keto reductase family 1, member B8

Gstp1 Akr7a3 Akr1b8

1.54 1.31 1.06

1.52 1.69 2.21

1.92 1.66 2.69

Growth arrest and DNA-damage-inducible, gamma DNA-damage-inducible transcript 4 Nitric oxide synthase 3, endothelial cell

Gadd45g Ddit4 Nos3

Data in bold indicate up-regulated expression (the criteria are shown in Section 2).

3.6. Gene expression analysis of testicular adhesion-related genes Since the GO analysis revealed that the cell adhesion-related genes were significantly enriched in the up-regulated genes by the DNB treatment, we further analyzed the expression changes of some of the cell adhesion-related genes, namely Cdh2, catenin alpha (Ctnna1), vinculin (Vcl), zyxin (Zyx), integrin beta 1 (Itgb1), Testin, laminin gamma 3 (Lamc3), poliovirus receptor-related 2 (Pvrl2, also known as nectin 2) and gelsolin (Gsn), as testicular cell junction related genes (Mruk and Cheng, 2004). As shown in Table 5, expressions of the selected genes showed a general tendency toward increase by DNB treatment. This increase in gene

expression levels of Testin and Cdh2 was also confirmed by the real-time RT-PCR (Fig. 5). 4. Discussion In the last decade, omics techniques have identified a number of novel biomarkers which provides toxicologists with profound insights into molecular mechanisms of drug-induced toxicities such as hepatotoxicity. The major objective of the present study

Table 3 Functional annotation of the up-regulated genes in DNB treated rats. GO ID

GO term

P-value

GO:0031589 GO:0048513 GO:0048731 GO:0030154 GO:0051239

Cell-substrate adhesion Organ development System development Cell differentiation Regulation of multicellular organismal process Phosphorus metabolic process Regulation of localization Regulation of cellular process Response to hormone stimulus Positive regulation of cell adhesion Anatomical structure morphogenesis Regulation of response to stimulus Response to organic substance Regulation of cell adhesion Positive regulation of biological process Regulation of system process Response to drug Anatomical structure formation involved in morphogenesis Response to hypoxia Response to inorganic substance Heterocycle metabolic process Response to oxygen levels Response to wounding Negative regulation of transport Positive regulation of immune response Regulation of developmental process Cell–cell adhesion Antigen processing and presentation of peptide antigen Regulation of immune response

4.98E−04 9.86E−04 1.01E−03 2.60E−03 3.03E−03

GO:0006793 GO:0032879 GO:0050794 GO:0009725 GO:0045785 GO:0009653 GO:0048583 GO:0010033 GO:0030155 GO:0048518 GO:0044057 GO:0042493 GO:0048646 GO:0001666 GO:0010035 GO:0046483 GO:0070482 GO:0009611 GO:0051051 GO:0050778 GO:0050793 GO:0016337 GO:0048002 GO:0050776

3.82E−03 1.05E−02 1.47E−02 1.65E−02 1.75E−02 1.76E−02 1.84E−02 1.89E−02 2.30E−02 2.44E−02 2.50E−02 2.82E−02 2.86E−02 2.91E−02 3.40E−02 3.52E−02 3.64E−02 3.74E−02 3.76E−02 3.84E−02 4.56E−02 4.77E−02 4.80E−02 4.85E−02

P-values were calculated using a modified Fisher Exact test in DAVID anotation system.

Fig. 5. Testicular gene expression changes of Testin and Cdh2 by real-time quantitative RT-PCR at 4 or 24 h after a single oral administration of DNB. Data show the mean ± SD of 4 animals. **P < 0.01: significantly different from the concurrent control group by Student’s t-test.

T. Matsuyama et al. / Toxicology 290 (2011) 169–177 Table 4 Functional anotation of the down-regulated genes in DNB treated rats. GO ID

GO term

P-value

GO:0007626 GO:0050794 GO:0048523 GO:0051171

Locomotory behavior Regulation of cellular process Negative regulation of cellular process Regulation of nitrogen compound metabolic process Taxis Chemotaxis Negative regulation of nitrogen compound metabolic process Leukocyte chemotaxis Negative regulation of biological process Cerebellum development Positive regulation of cell proliferation Cell chemotaxis Metencephalon development

7.79E−03 1.08E−02 1.52E−02 2.47E−02

GO:0042330 GO:0006935 GO:0051172 GO:0030595 GO:0048519 GO:0021549 GO:0008284 GO:0060326 GO:0022037

3.19E−02 3.19E−02 3.71E−02 3.79E−02 3.83E−02 3.98E−02 4.31E−02 4.37E−02 4.98E−02

P-values were calculated using a modified Fisher Exact test in DAVID anotation system.

was to identify novel toxicogenomic biomarkers which add values to conventional biomarkers for evaluation of testicular toxicity. For this purpose, we used DNB to generate a chemical-induced testicular toxicity rat model. The histopathology examination clearly demonstrated morphological changes in spermatocytes and Sertoli cells, namely, single death of spermatocytes and Sertoli cell vacuolation were observed in testis at 25 and 50 mg/kg 24 h after single treatment of DNB. These were known to be major pathological findings induced by a single dose of DNB as reported in previous papers (Blackburn et al., 1988; Hess et al., 1988). Using this testicular toxicity rat model, we first evaluated the usefulness of the cell type-specific gene sets whose expression levels are exclusively greater in one of the SC-, PS-, SG- and RS-cells. These cell type-specific gene sets were determined by modifying the contents of the gene sets by selecting genes whose expression levels are greater than 4-fold compared to other types of cells, based on the previously published report (Johnston et al., 2008). Using these cell type-specific gene sets, our microarray data on rat testis following DNB treatment clearly demonstrated that PS-specific genes elicited marked down-regulations, while there was little effect on expression levels of SC-, SG- and RS-specific genes. Down-regulation of PS-specific genes was thought to reflect progression of single death of spermatocytes following DNB treatment. These findings demonstrate the strong correlation between expression change in PS-specific genes and spermatocytes injury, suggesting that the PS-specific gene sets used in the present study

175

can be used for evaluating drug-induced testicular toxicity targeting pachytene spermatocytes. Although histopathology examination is a straightforward way to evaluate the drug-induced germ cell injuries, the damaged germ cells could rapidly disappear due to phagocytosis by the Sertoli cells or by exfoliation into the tubular lumen (Creasy, 1997), and therefore it is not always easy to appropriately evaluate the germ cell injury only by histopathology examination. On the other hand, considering that the down-regulation of PS-specific gene expression was thought to reflect the decrease in the number of spermatocytes, scoring the degree of down-regulation of the PS-specific genes would be a convenient biomarker to evaluate PS-specific cell injury. In other words, scoring the PS-specific gene expression profiling possesses the advantages of being more sensitive, objective and quantitative compared with a conventional histopathology examination. Next, we examined the list of differentially regulated genes in the testis by DNB treatment. In the present study, we focused on the genes which were differentially regulated at 24 h where histological changes were observed, because such phenotypically anchored analysis methods can provide us with reliable and objective interpretation of toxicogenomic data. Previous studies suggested that DNB induces oxidative stress (Jacobson and Miller, 1998; Reeve et al., 2002) and apoptosis through a mitochondrial pathway (Muguruma et al., 2005). In the present study, oxidative stress-related genes exhibited significant up-regulations at 24 h after the DNB treatment. For example, genes responsive to oxidative stress, namely, Hmox1 and Pon2 (Devarajan et al., 2011; Guo et al., 2001) and protective against oxidative stress, namely, Gstp1, Akr7a3 and Akr1b8 (Ahmed et al., 2011; Ikeda et al., 2004; Jin and Penning, 2007) were up-regulated in the DNB-treated rat testis (Table 2), supporting the information from previous reports that DNB induces oxidative stress in the rat testis. In addition, DNA damage-responsive genes Gadd45g and Ddit4 (Linschooten et al., 2011; Miyake et al., 2007), which will lead to apoptosis, showed significant up-regulations. Furthermore, Nos3 was up-regulated in the DNB-treated rat testis. Since the elevation in NO production could induce apoptosis and may impair spermatogenesis (Taneli et al., 2005), up-regulation of Nos3 may be associated with DNB-induced cellular apoptosis in the testis. These data also support the information that apoptosis would be associated with DNB-induced testicular toxicity in the rat. However, genes reported to be up-regulated in the DNB-treated rat testis such as Bcl2, Bcl2l1 (Bcl-xL), and Casp6 did not change in their gene expression levels in the present study, which may be due to weak toxicity level. These data implies that oxidative stress would be one

Table 5 Cell adhesion related genes. Probe ID

Gene expression (/control) 4h

1368642 1387259 1371921 1372905 1375538 1398476 1388130 1368819 1387346 1370457 1393896 1375216 1371414

at at at at at at at at at at at at at

Gene title

Gene symbol

Cadherin 2 Cadherin 2 Catenin (cadherin associated protein), alpha 1 Vinculin Vinculin Vinculin Zyxin Integrin beta 1 (fibronectin receptor beta) Integrin beta 1 (fibronectin receptor beta) Testin gene Laminin gamma 3 Poliovirus receptor-related 2 Gelsolin

Cdh2 Cdh2 Ctnna1 Vcl Vcl Vcl Zyx Itgb1 Itgb1 Testin Lamc3 Pvrl2 Gsn

24 h

25 mpk

50 mpk

25 mpk

1.01 1.06 0.96 0.96 1.08 0.91 1.08 0.98 0.99 1.25 0.97 1.01 0.84

1.32 1.15 0.96 0.90 1.07 0.85 1.09 1.05 0.99 1.42 1.03 1.07 0.89

1.70 1.58 1.41 1.23 1.28 1.09 1.32 1.48 1.36 2.22 1.22 1.55 1.15

Bold indicate statistically significant changes (Student’s t test, P ≤ 0.05).

50 mpk 2.88 3.73 1.72 1.75 1.95 1.77 1.76 2.11 2.08 5.47 1.45 1.60 1.37

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of the early events caused by DNB, followed by cell death including apoptosis. To gain insight into novel molecular mechanisms caused by DNB treatment, we conducted a Gene Ontology analysis on a differentially regulated gene list. The GO analysis revealed characteristic up-regulations of cell adhesion-related genes in the DNB-treated rat testis, and the up-regulations of Cdh2 and Testin genes at 24 h were confirmed by real time RT-PCR analysis. Cdh2, also known as N-cadherin, is reported to localize at basal inter-Sertoli junctions, Sertoli–spermatocyte junctions, and Sertoli–elongate spermatid junctions (Johnson and Boekelheide, 2002). Up-regulation of Cdh2 in testis was also observed in rats treated with di-(2-ethylhexyl) phthalate, which is a typical Sertoli cell toxicant and causes disruption of seminiferous tubules with sloughing of germ cells (Sobarzo et al., 2006). Up-regulation of the testicular Cdh2 gene was also observed in the rats treated with AF-2364, a male contraceptive that induces germ cell loss from the seminiferous epithelium via disruption of Sertoli–germ cells adherens junctions (Lee et al., 2003). Thus, the up-regulation of Cdh2 gene in the DNB-treated rat testis may indicate loss of Sertoli–germ cells adhesion. On the other hand, Testin is a secretory protein from Sertoli cells (Cheng et al., 1989) and localized on the Sertoli cell side of the ectoplasmic specialization surrounding developing spermatids (Grima et al., 1998). Expression of Testin relates only to the disruption of Sertoli–germ cells junctions, therefore, induction of the Testin expression appears to be a indicator for monitoring the loss of integrity of inter-testicular cell junctions (Grima et al., 1997). Oral administration of lonidamine, a male contraceptive agent causing disruption of Sertoli–germ cells adhesion junction, induced an increase in testicular Testin expression (Grima and Cheng, 2000). In general, disruption of Sertoli–germ cells contacts cause germ cell sloughing from seminiferous epithelium (Richburg et al., 1997). Several studies have shown that DNB induces germ cell sloughing in in vivo and in vitro (Foster et al., 1987; Hess et al., 1988). In our study, increased debris of the spermatogenic cell in epididymis was observed at 24 h after DNB treatment. This finding suggested a disruption of Sertoli–germ cells junctions by the DNB treatment, resulting in a loss of cell adhesion. Taken together, up-regulation of cell adhesion-related genes including Cdh2 and Testin observed in this study suggests disruption of Sertoli–germ cells adhesion by DNB treatment, which might be a consequence of cellular response for Sertoli–germ cells adhesion restructuring. Thus, the up-regulation of testicular cell adhesion-related genes such as Cdh2 and Testin could be potential biomarkers to evaluate such a cellular response elicited in the DNB-type testicular toxicity. In addition, significant up-regulation of Cdh2 and Testin were detected by microarray analysis at 4 h, where no histological change was observed. Although these expression changes were slight and could not be confirmed by real time RT-PCR analysis, these results may imply that these expression changes detected by microarray analysis might be early signs for disruption of Sertoli–germ cells adhesion before the histological changes occur. In conclusion, we confirmed characteristic expression changes of PS-specific genes and cell adhesion-related genes in DNB-treated rat testis, which were thought to reflect the decrease of spermatocytes and dysfunction of Sertoli–germ cells adhesion junction elicited by DNB treatment. Since the magnitude of expression change of these genes are thought to be associated with the progression of testicular toxicity, expression profiles of these gene sets would be useful toxicogenomic biomarkers for assessing DNB-type testicular toxicity.

Conflict of interest statement The authors declare that there are no conflicts of interest.

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