Maternal exposure to benzo[b]fluoranthene disturbs reproductive performance in male offspring mice

Toxicology Letters 203 (2011) 54–61

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Maternal exposure to benzo[b]fluoranthene disturbs reproductive performance in male offspring mice Ahyoung Kim a , Mira Park b , Tae Ki Yoon c , Woo Sik Lee c , Jeong-Jae Ko a , Kangseok Lee d , Jeehyeon Bae b,∗ a

Department of Biomedical Science, College of Life Science, CHA University, Seongnam 463-836, Republic of Korea Department of Pharmacy, College of Pharmacy, CHA University, Seongnam 463-836, Republic of Korea Fertility Center of CHA Gangnam Medical Center, Seoul 135-081 Republic of Korea d Department of Life Science, College of Natural Science, Chung-Ang University, Seoul 156-756, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 20 January 2011 Received in revised form 26 February 2011 Accepted 2 March 2011 Available online 8 March 2011 Keywords: Endocrine disruptor Polycyclic aromatic hydrocarbon Testis Apoptosis Estrogen receptor Sperm quality

a b s t r a c t Polycyclic aromatic hydrocarbons (PAHs) are a large family of environmentally prevalent toxic compounds generated from the combustion of organic materials and diesel exhaust. Humans and wild animals are exposed to PAHs mostly through dietary intake of contaminated food. Benzo[b]fluoranthene (B[b]F) is a common constituent of PAH complexes present in diverse types of food. B[b]F has been found in human milk, raising the demand for the need for risk assessment of offspring after maternal exposure to B[b]F. In the present study, pregnant mice were orally exposed to low doses (2–2000 ␮g/kg body weight) of B[b]F during gestational and lactational periods, and their male offspring were assessed. Maternal B[b]F exposure disturbed normal sperm function in F1 offspring. To understand the molecular and cellular mechanisms by which the perinatal exposure to B[b]F decreased sperm quality, the testes of young adult F1 mice were examined for changes in expression of steroidogenesis-related and testicular apoptosis mediators and found that aryl hydrocarbon receptor, estrogen receptor ␣, and a set of proapoptotic proteins including Bax, Noxa, Bad, and Bim were significantly upregulated. Therefore, the current transgenerational animal study implies that consumption of PAH-contaminated diets by mothers may possibly influence their offspring to cause dysfunctional male reproductive function in humans. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are a large family of toxic compounds generated from the combustion of organic materials, diesel exhaust, and industrial waste by-products, and they are widely spread pollutants present in the atmosphere, water, and soil. Humans and wild animals are exposed to a diverse array of PAHs, and the primary source of PAH exposure in the general population is through dietary intake of contaminated food (Gilbert, 1994). Some PAHs are classified as human carcinogens (IARC, 2010) and also elicit a broad spectrum of toxic responses in human and animals. Exposure to a highly toxic PAH, benzo[a]pryene (B[a]P), caused infertility in mice (MacKenzie and Angevine, 1981; Mattison and Nightingale, 1982) and subcutaneous administration of B[a]P, benzo[a]anthracene, or fluoranthene in immature rats produced estrogenic responses (Kummer et al., 2008). Benzo[b]fluoranthene (B[b]F), a non-alternant PAH congener, is a common constituent of PAH complexes produced from the incomplete combustion or pyrolysis of organic materials and is designated as a probable

∗ Corresponding author. Tel.: +82 31 725 8396; fax: +82 31 725 7350. E-mail address: [email protected] (J. Bae). 0378-4274/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2011.03.003

human carcinogen based on IRIS (Integrated Risk Information System) (US EPA 1999). Despite the prevalent presence of B[b]F in foods, such as meat, fish, shellfish, vegetables, fruits, eggs, dairy products, cereals, and oils (Martorell et al., 2010; The Annual Report of KFDA 2004), most studies have focused on its carcinogenic activity at higher doses, and scarce data are available concerning its reproductive toxicities. The testis is the central organ in the male reproductive system and is composed of the seminiferous tubules and the interstitial space between the tubules. Spermatogenesis is a dynamic and complex process leading to testicular germ cell development in which the total balance of estrogen and androgen is required (Carreau et al., 1998; O’Donnell et al., 2001). The quality of human semen over a 50-year period has declined (Carlsen et al., 1992), and epidemiology studies suggest that environmental exposures are associated with the decline in sperm quality (Jurewicz et al., 2009; Swan, 2006). B[b]F has been detected in the milk of healthy, nonsmoking, lactating women at 0.560 ± 1.39 ␮g/kg milk as wet weight (Del Bubba et al., 2005), raising the demand for the need for risk assessment of offspring after maternal exposure to B[b]F. In the present study, pregnant mice were orally exposed to low doses of B[b]F, at which no signs of obvious maternal toxicity were observed, during gestational and lactational periods, and their male offspring

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were assessed. We found that maternal B[b]F exposure decreased the sperm quality and dysregulated the expression of critical proteins involved in steroidogenesis and apoptosis signaling in their offspring. 2. Materials and methods 2.1. Chemicals Benzo[b]fluoranthene (CAS # 205-99-2; 98% pure) was purchased from the Supelco (Bellefonte, PA, USA), and other chemicals were obtained from Sigma (St Louis, MO, USA) unless otherwise indicated. 2.2. Animals Eleven-week-old C57BL/6 female and seven-week-old male mice were obtained from SLC Inc. (Tokyo, Japan) and mated to obtain F1 offspring. The animal room was maintained at a humidity of 30–40% and a temperature of 22 ± 1 ◦ C. Lighting in the room was on a 12-h light/dark cycle. All animals were given water and AIN76A rodent feed ad libitum (Research Diets, New Brunswick, NJ, USA). The animals were treated humanely and to ease suffering according to the experimental protocol approved by CHA University Institutional Animal Care and Use Committee. Female mice were orally administered by B[b]F (0, 20, 200 or 2000 ␮g/kg body weight) by gavage daily in 0.1 mL corn oil from gestation day (GD) 12–16 to postnatal day (PND) 2–21 (Fig. 1). 2.3. Animal assessment Mice of PND 14 and 21 and 8–9-week-old male offspring (F1) and their dams (F0) were euthanized by cervical dislocation; the body and organ weights were measured. Collected tissue samples were submerged either in RNAse-free water pre-treated with 0.1% (v/v) diethyl pyrocarbonate (DEPC) and then snap-frozen in liquid nitrogen gas or in 4% formaldehyde (Sigma, Steinheim, Germany) and stored in 4 ◦ C. The anogenital distance (AGD) of offspring was measured on PND 14 and 21. AGD was measured from the centre of the anus to the posterior convergence of the fourchette in female mice and from the centre of the anus to the junction of the smooth perineal skin with the rugated skin of the scrotum in male mice. Also, the AGD index, which is the ratio of AGD to the cube root of body weight, was calculated as a more accurate measurement of AGD (Gallavan et al., 1999). 2.4. Computer assisted sperm analysis (CASA) The cauda distal epididymides were punctured in a 60-mm organ culture dish (Orange Scientific Inc., Braine-I’Alleud, Belgium) containing 1.0 mL of pre-warmed Dulbecco’s modified Eagle medium with Ham’s F-12 nutrient mixture (DMEM/F12; Welgene, Seoul, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Welgene). Sperm concentration and motion analyses were conducted within 30 min after puncture. Quantitative parameters of sperm motility were measured by CASA using the integrated visual optical system (IVOS) motility analyzer (Hamilton-Thorne Research Inc., Beverly, MA, USA), software version 10.7. Quantitative parameters of sperm motility evaluated in this study were the standard mouse parameters as recommended by the manufacturer: sperm concentration, % motility (MOT), track velocity (VCL), the sum of the distances between each centre of brightness, during each frame, divided by the time elapsed; straight line velocity (VSL), the distance between the first and last tracked point of the spermatozoon trajectory divided by the time elapsed; smoothed path velocity (VAP), a smoothing of the path of the centre of brightness of the spermatozoon, which reduces the effect of the lateral head displacement; linearity (LIN), measures the straightness of the path (the ratio of VSL/VCL multiplied by 100); amplitude of lateral head displacement (ALH), the maximum value of the distance of any point on the track from the corresponding average path, multiplied by two; straightness (STR), measures the departure of the cell path from a straight line (the ratio of VSL/VAP multiplied by 100); beat cross frequency (BCF), the frequency with which the cell track crosses the cell path in either direction; mean angular displacement (MAD); and wobble coefficient (WOB), curvilinear progressiveness ratio (the ratio of VAP/VCL multiplied by 100).

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2.6. Immunohistochemistry Paraffin-embedded testes were cut at 5 ␮m and deparaffinized. The sections were rehydrated, immersed in an antigen retrieval solution (0.01 M sodium citrate, 0.05% Tween 20, pH 6.0), and microwaved for 10 min (100 ◦ C at 600 W). Endogenous peroxidase was blocked in 3% hydrogen peroxidase solution (Duksan) for 10 min and rinsed in phosphate-buffered saline (PBS). Nonspecific staining was blocked with 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. The sections were then incubated with polyclonal anti-ER␣ (1:100), androgen receptor (AR) (1:100), aryl hydrocarbon receptor (AhR) (1:100), Bax (1:50), Bak (1:50), Noxa (1:50), Bad (1:50), or Bim (1:30) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) in the antibody diluent (Dako, Carpinteria, CA, USA) for 20 h at 4 ◦ C. For the incubation with secondary antibody, the Universal LSABTM + system kit (Dako) was used as suggested by the manufacturer. All sections were counterstained with Mayer hematoxylin for 5 min. Negative controls were used for each probe by substituting the antibody with 2.5% normal horse serum (Vector Laboratories, Burlingame, CA, USA).

2.7. Western blot analysis Lysates were prepared from testes of 8-week-old offspring using PRO-PREP solution (Intron, Seongnam, Korea) following homogenization. Equal amounts of total protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to membranes. The membranes were immunoblotted with anti-ER␣, and the same membranes were incubated with ␤actin (Ab Frontier, Seoul, Korea). After washing, membrane blots were incubated at room temperature for 2 h with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody at a 1:5000 dilution (Santa Cruz Biotechnology) before visualization using enhanced chemiluminescence (AB Frontier, Seoul, Korea) and detected using LAS3000 Intelligent Dark Box Imager (Fuji, New York, NY, USA). Relative expression (%) was calculated based on protein densities with MultiGauge V 3.0 software (Fuji).

2.8. Measurement of androstenedione Plasma of 8-week-old offspring was purified, and an androstenedione ELISA (enzyme-linked immunosorbent assay) kit was used following the manufacturer’s instructions (Neogen, Lexington, KY, USA). Briefly, the standard curve was generated, and androstenedione from the plasma was extracted with ethyl ether and the extraction buffer provided. Each well was incubated with the androstenedione enzyme conjugate diluted in the EIA buffer followed by washings. The substrate was then added to the wells and incubated at room temperature for 30 min. The absorbance was measured using a Perkin-Elmer 1420 Multilabel Counter (PerkinElmer, Waltham, MA, USA) at 650 nm.

2.9. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) DNA fragmentation in the testes of 8-week-old mice was assessed in situ with TUNEL (Roche, Mannheim, Germany) as described by the manufacturer, with minor modifications. Paraffin sections of 5-␮m thickness were treated with proteinase K (20 ␮g/mL) for 15 min at room temperature after blocking of the endogenous peroxidase activity with 1% hydrogen peroxide for 10 min. After washing in 0.01 M PBS, the slides were incubated in TUNEL Mix comprising 0.01 U/␮L terminal transferase, 1 mM/L CoCl, 0.4 mM/L digoxigenin deoxyuridine triphosphate, and reaction buffer (200 mM/L KCl, 25 mM/L Tris–HCl, 0.25 mg/mL bovine serum albumin, pH 6.6) for 1 h at 37 ◦ C. The reaction was terminated by washing in 0.2% saline-sodium citrate (SSC; 3 M NaCl, 0.3 M sodium citrate, pH 7.4). The sections were then treated with anti-digoxigenin peroxidase-conjugated sheep polyclonal antibody (1:500) for 30 min at 37 ◦ C. Slides were washed in PBS, and color was developed using 3,3 -diaminobenzidine (DAB). The slides were then counterstained with hematoxylin. The cells exhibiting brown staining were considered to be positive for apoptosis-associated DNA fragmentation. Negative controls were processed in a similar manner, with the omission of terminal transferase in the reaction mixture.

2.10. Statistical analysis 2.5. Histological analysis of testes The testes from 8 to 9-week-old F1 male mice were immersion-fixed in 4% neutral buffered formalin and then stored at 4 ◦ C until analysis. The tissues were transferred to 70%, 80%, 90%, 95%, and 100% ethanol (Merck, Darmstadt, Germany) for 1 h each to dehydrate and then were transferred to a xylene (Duksan, Ansan, Korea) tank for clearing. The tissues were embedded in paraffin, sliced at intervals of 5 ␮m (Thermo Scientific, Rockfold, IL, USA), and placed on slides. At room temperature, slides were deparaffinized and stained with hematoxylin and eosin (H&E).

Data analysis was performed using SAS version 8.0 (SAS Institute, Cary, NC, USA). All data were tested for normality by the Shapiro–Wilk test at the 5% level of significance. Non-normal data were log transformed and retested. Nonparametric one-way ANOVA using the NPAR1WAY procedure of SAS was performed for nonnormal data. The Kruskal–Wallis test was used to compare the control and the tested groups. Data that passed the normality test were analysed with a repeated measures ANOVA using the PROC GLM procedure of SAS. To compare the control and the treated groups, Dunnett’s and Student’s t-tests were performed. The level of significance was set at p < 0.05.

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Fig. 1. A scheme of the in vivo animal experiment. Dams (F0) were mated, and pregnant mothers were exposed to different daily doses of B[b]F from gestational day (GD) 12–16 to postnatal day (PND) 2–21. The assessment of body and organ weights, anogenital distance (AGD), computer assisted sperm analysis (CASA), tunnel assay, immunohistochemistry, and western blot of their male offspring (F1) was performed at the indicated time points.

Table 1 Body weight and anogenital distance of F1 male mice exposed to B[b]F maternally. Maternal doses of B[b]F (␮g/kg/day) Number of animals (# of litter/# of dam) Male body weight (g) PND 14 PND 21 Male AGD (mm) PND 14 PND 21 Male AGDa PND 14 PND 21

0 47/11

2 41/10

20 34/9

200 34/9

2000 38/9

5.90 ± 0.09 7.43 ± 0.15

6.10 ± 0.12 7.77 ± 0.20

6.45 ± 0.11*** 7.94 ± 0.21*

6.08 ± 0.12 7.63 ± 0.20

6.27 ± 0.14* 8.22 ± 0.23***

2.31 ± 0.06 3.45 ± 0.09

2.52 ± 0.07* 3.65 ± 0.13

2.26 ± 0.05 3.79 ± 0.05*

2.24 ± 0.06 3.89 ± 0.11**

2.78 ± 0.10*** 4.00 ± 0.16**

1.28 ± 0.03 1.77 ± 0.04

1.38 ± 0.04* 1.84 ± 0.05*

1.22 ± 0.03 1.91 ± 0.03***

1.23 ±0.03 1.98 ± 0.03***

1.51 ± 0.05*** 1.99 ± 0.03**

All values are means ± SEM. Significantly different from control; p < 0.05*, p < 0.005**, p < 0.0005***. a AGD/cube root of body weight.

Table 2 Epididymal sperm concentration and quality of young adult F1 offspring exposed to B[b]F. Maternal doses of B[b]F (␮g/kg/day) Number of animals

0 11

Sperm concentration (106 /mL) MOT (% Motility) VCL (␮m/s) VSL (␮m/s) VAP (␮m/s) LIN (%) ALH (␮m) STR (%) BCF (Hz) MAD WOB

42.26 ± 7.14 52.31 ± 4.24 52.28 ± 3.90 14.83 ± 0.84 28.09 ± 2.09 43.56 ± 6.00 1.25 ± 0.10 60.90 ± 7.86 5.75 ± 0.31 31.96 ± 1.96 60.91 ± 6.34

2 7

20 6

32.70 ± 5.73 53.34 ± 4.18 44.27 ± 2.91 14.04 ± 0.66 24.36 ± 1.54 42.31 ± 2.30 1.09 ± 0.07 58.74 ± 2.54 4.94 ± 0.20* 29.49 ± 1.41 56.41 ± 1.46

30.18 ± 4.46 36.40 ± 9.89 32.25 ± 5.44 11.12 ± 1.49 18.45 ± 3.09* 46.12 ± 2.22 0.78 ± 0.14* 62.87 ± 3.20 4.05 ± 0.41* 24.65 ± 2.46 58.43 ± 1.20

200 11 44.15 ± 7.18 40.75 ± 4.59 40.15 ± 2.63* 12.15 ± 0.40* 21.66 ± 1.33* 42.60 ± 1.73 0.95 ± 0.07* 57.86 ± 2.00 4.27 ± 0.34* 25.19 ± 2.57 55.61 ± 1.27

2000 8 66.05 ± 11.10 43.10 ± 5.49 43.41 ± 2.21 12.35 ± 0.87 22.93 ± 1.25 39.28 ± 2.91 1.05 ± 0.04 53.16 ± 3.87 4.04 ± 0.46* 20.75 ± 2.32** 51.70 ± 3.35

All values are means ± SEM. MOT: motility; VCL: track velocity; VSL: straight line velocity; VAP: smoothed path velocity; LIN: linearity; ALH: amplitude of lateral head displacement; STR: straightness; BCF: beat cross frequency; MAD: mean angular displacement; WOB: wobble coefficient. Significantly different from control; p < 0.05*, p < 0.005**.

3. Results 3.1. Altered body weight and anogenital distance of male offspring exposed to benzo[b]fluoranthene Daily oral exposure of C57BL/6 female mice to B[b]F at 2, 20, 200, and 2000 ␮g/kg body weight from GD 12 to PND 24 (Fig. 1) did not cause any obvious toxic effects on dams (F0); weight gain and reproductive performance between groups were not significantly different (Supplementary Table 1), suggesting that the doses used in the current study were not high enough to elicit mater-

nal toxicity. In contrast, the maternal exposure to B[b]F led to an increased average body weight of their male offspring (F1) at PND 2, and the body weight gain effect was also observed at PND 14 and 21 (Table 1 and Supplementary Table 1). In addition, the AGD on PND 14 or 21 of the male F1 mice groups exposed to 2 and 2000 ␮g/kg B[b]F or to 20, 200, and 2000 ␮g/kg B[b]F, respectively, was significantly lengthened by 9.10–12.03% of the AGD of the control group (Table 1). Although the AGD of the offspring on PND 21 showed dose-dependent lengthening, PND 14 offspring did not showed the dose-dependency of which the reason is not clear at this point.

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Table 3 Body and organ weights of young adult F1 male mice exposed to B[b]F. Maternal doses of B[b]F (␮g/kg/day) Number of animals

0 11

Body weight (g)

24.97 ± 0.57

Absolute organ weights (mg) Paired Testis Paired Epididymis Seminal vesicle Paired Kidney Spleen Liver Heart Lung Thymus gland Adjusted organ weight (mg/g)a Paired Testis Paired Epididymis Seminal vesicle Paired Kidney Spleen Liver Heart Lung Thymus gland

2 8

20 6

25.34 ± 0.38

26.20 ± 0.70

200 11 23.96 ± 1.47

2000 8 25.20 ± 0.92

0.1830 ± 0.0064 0.0672 ± 0.0053 0.2222 ± 0.0145 0.4271 ± 0.0340 0.1076 ± 0.0107 0.9229 ± 0.0767 0.1501 ± 0.0097 0.1639 ± 0.0055 0.0515 ± 0.0044

0.1892 ± 0.0089 0.0742 ± 0.0068 0.2313 ± 0.0293 0.3844 ± 0.0447 0.0913 ± 0.0106 1.0109 ± 0.1299 0.1421 ± 0.0085 0.1642 ± 0.0073 0.0671 ± 0.0138

0.1980 ± 0.0069 0.0701 ± 0.0032 0.2905 ± 0.0209* 0.4207 ± 0.0235 0.0849 ± 0.0057 1.0913 ± 0.1101 0.1478 ± 0.0051 0.1630 ± 0.0037 0.0445 ± 0.0024

0.1755 ± 0.0086 0.0631 ± 0.0040 0.2214 ± 0.0209 0.3207 ± 0.0220* 0.0684 ± 0.0050* 1.1477 ± 0.0857 0.1275 ± 0.0043 0.1509 ± 0.0075 0.0401 ± 0.0033*

0.1768 ± 0.0041 0.0660 ± 0.0021 0.2255 ± 0.0132 0.3247 ± 0.0123 0.0706 ± 0.0030* 1.2741 ± 0.0246** 0.1339 ± 0.0046 0.1558 ± 0.0089 0.0548 ± 0.0094

0.7398 ± 0.0252 0.2718 ± 0.0206 0.9028 ± 0.0573 1.6843 ± 0.1327 0.4232 ± 0.0468 3.8276 ± 0.3284 0.5868 ± 0.0360 0.6469 ± 0.0189 0.2049 ± 0.0216

0.7449 ± 0.0276 0.2920 ± 0.0249 0.9057 ± 0.1067 1.5125 ± 0.1733 0.3623 ± 0.0447 3.9787 ± 0.5039 0.5609 ± 0.0337 0.6474 ± 0.0255 0.2632 ± 0.0536

0.7578 ± 0.0300 0.2674 ± 0.0099 1.1024 ± 0.0601* 1.6206 ± 0.1329 0.3282 ± 0.0324 4.1215 ± 0.3383 0.5678 ± 0.0336 0.6253 ± 0.0279 0.1717 ± 0.0141

0.7523 ± 0.0431 0.2647 ± 0.0119 0.8999 ± 0.0676 1.3361 ± 0.0414 0.2830 ± 0.0139* 4.8209 ± 0.2304* 0.5486 ± 0.0309 0.6431 ± 0.0309 0.1594 ± 0.0107*

0.7066 ± 0.0246 0.2632 ± 0.0083 0.8946 ± 0.0406 1.2956 ± 0.0520 0.2801 ± 0.0053* 5.0971 ± 0.1876* 0.5355 ± 0.0234 0.6265 ± 0.0429 0.2163 ± 0.0337

All values are means ± SEM. Significantly different from control; p < 0.05*, p < 0.005**. a (Organ weight/body weight at the time of necropsy) × 100.

Fig. 2. Testicular histology of F1 offspring exposed to B[b]F. (A) H&E stained histology of testicular cross-sections of 8–9-week-old offspring maternally exposed to corn oil (a and b) and 20 ␮g/kg (c and d), 200 ␮g/kg (e and f) and 2000 ␮g/kg B[b]F (g and h) is shown. (B) Immunohistochemical analysis of F1 testes described in (A) with anti-AhR antibody is presented; (a) corn oil (b) 20 ␮g/kg (c) 200 ␮g/kg and (d) 2000 ␮g/kg B[b]F.

3.2. Decreased sperm quality of the male offspring exposed to B[b]F To determine the reproductive performance of the offspring maternally exposed to B[b]F, epididymal sperm of 8–9-week-old

F1 mice was collected, and CASA was performed. Sperm concentrations of mice groups exposed to different doses of B[b]F were not significantly different compared to the control (Table 2). However, the parameters of sperm quality, such as VCL, VSL, VAP, ALH, BCF, and MAD, were significantly decreased, while MOT, LIN, STR,

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Fig. 3. Upregulated ER␣ expression in B[b]F-exposed male offspring. (A) The testis sections of young adult F1 male mice as described in Fig. 2A were immunostained for ER␣; (a and b) corn oil, (c and d) 20 ␮g/kg, (e and f) 200 ␮g/kg, and (g and h) 2000 ␮g/kg B[b]F. (B) Proteins were isolated from the testes of F1 mice exposed to different doses of B[b]F. Western blot analysis was conducted using anti-ER␣ antibody; a representative blot is shown (upper level). Equal loading of each sample was confirmed by immunoblotting of the same membrane using anti-␤-actin antibody. Data (lower level) are mean ± SEM from seven independent testes in each dose of B[b]F and presented as % ER␣ expression level normalized by ␤-actin. Asterisks indicate statistically significant value relative to the control, 0 ␮g/kg/day (p < 0.05).

and WOB were not significantly affected (Table 2). BCF, which is the frequency with which sperm crosses the smoothed path, was decreased in all the groups of B[b]F-exposed mice ranging from 2 to 2000 ␮g/kg (Table 1), suggesting the motility of sperm was greatly affected in the offspring exposed to B[b]F.

observe major differences in gross anatomical morphology of the seminiferous tubules between the exposed and the control groups (Fig. 2A). 3.5. Increased AhR and ER˛ expression in the testes of B[b]F-exposed F1s

3.3. Effects of B[b]F on organ weights of F1 male mice The body weights of 8–9-week-old F1 male offspring that were analysed for CASA were not significantly different between groups but certain organ weights were different as shown in Table 2. The weight of the seminal vesicle was increased in the 20 ␮g/kg B[b]F group, whereas testicular and epididymal weights were not changed (Table 3). The weights of kidney, spleen, and thymus gland of B[b]F-exposed groups were significantly decreased, while liver weight was increased notably (Table 3). The changes in some of the organ weights were consistently observed even after adjustment for body weight, including increased weight of the seminal vesicle and liver and decreased weight of the spleen and thymus gland (Table 3). 3.4. The testicular change in the testes of the F1s exposed to B[b]F Because the sperm quality was diminished in the B[b]F-exposed mice, we next performed histological analyses of testes of F1 young adult offspring mice to compare any distinctive changes. We did not

Because the B[b]F-exposed mice showed increased AGD (Table 1) and decreased sperm quality (Table 2), we then examined if B[b]F exposure led to alteration in the expression of AhR and sex hormone receptors. As shown in Fig. 2B, AhR expression was intensified in B[b]F groups. Also, the male offspring exposed to B[b]F expressed increased ER␣ in Sertoli, germ, and Leydig cells within the seminiferous tubules (Fig. 3A). In addition, significantly upregulated ER␣ expression in the protein extract of the testes of B[b]F-exposed groups was confirmed by western blot analysis (Fig. 3B). In contrast, expression patterns of AR, Cyp17, and Cyp19 and the level of serum androstenedione were not significantly altered in the testes of the offspring after the maternal exposure to B[b]F (Supplementary Fig. 1A–D) 3.6. Increased testicular apoptosis in the offspring exposed to B[b]F In addition, we determined whether perinatal exposure to B[b]F increased testicular apoptosis. The TUNEL-positive cells with

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Fig. 4. Increased testicular apoptosis of offspring exposed to B[b]F. (A) The TUNEL assay was conducted on the cross-sections of adult male testes prepared from groups exposed to corn oil (c) or 20 ␮g/kg (d), 200 ␮g/kg (e), or 2000 ␮g/kg B[b]F (f). DNase I was treated for the TUNEL-positive control (a), and the TdT enzyme was not treated for the negative control (b). The cross-sections of F1 testes were immunostained for Bax (B) and Bak (C); (a) corn oil, (b) 20 ␮g/kg, (c) 200 ␮g/kg, and (d) 2000 ␮g/kg B[b]F. Immunohistochemical analyses of Noxa (D), Bad (E), and Bim (F) are presented using the testicular cross-sections of corn oil-fed control (a) and 200 ␮g/kg B[b]F-exposed F1 offspring (b).

brownish staining clearly increased in B[b]F groups especially for spermatocytes (Fig. 4A), suggesting that the maternal exposure to B[b]F resulted in testicular germ cell apoptosis in the F1 offspring. Because Bcl-2 family proteins are critical members that regulate cell death of diverse species (Dewson et al., 2008), we next assessed if expression of any particular members of the proapoptotic Bcl2 family were affected by B[b]F exposure. Immunohistochemical analyses of testes from offspring for Bax, Bak, Noxa, Bad, and Bim were performed. The expression level of Bax was increased in testicular germ cells in a B[b]F dose-dependent manner, while the level of its close homologue Bak did not show any significant changes (Fig. 4B and C). In addition, the levels of BH3-only members of the proapoptotic Bcl-2 family, Noxa, Bad, and Bim, increased mainly in Leydig cells of the 200 ␮g/kg B[b]F-exposed group (Fig. 4D–E). 4. Discussion In the present study, we found that male offspring of mice maternally exposed to relatively low doses of B[b]F during critical periods had different toxic responses including dysregulated sperm function. To understand the molecular and cellular mechanisms by

which the perinatal exposure to B[b]F decreased sperm quality, the testis of young adult mice exposed to B[b]F maternally were examined for changes in expression of steroidogenesis-related receptors and testicular apoptosis, and we found that AhR, ER␣, and a set of proapoptotic proteins were significantly upregulated. AhR is a ligand-activated transcriptional factor that mediates diverse biological responses of PAHs upon its translocation to the nucleus. We found that the expression level of AhR in testicular germ cells of B[b]F-exposed offspring was increased in a dosedependent manner (Fig. 2B), indicating that maternal exposure to B[b]F upregulates AhR expression in the germ cells of F1 mice. AhR protein is known to be expressed in all types of cells in the seminiferous tubules and interstitial cells of human testis (Schultz et al., 2003), and its altered expression in a rat Leydig cell line after B[a]P treatment in vitro has been reported (Chung et al., 2007). In this study, we also found that ER␣ level was significantly increased in the testis of B[b]F-exposed F1 mice (Fig. 3). Although the crosstalk between AhR and ER occurs via multiple mechanisms (Swedenborg and Pongratz, 2010; Ohtake et al., 2007), it is unknown at this point if ER␣ upregulation observed in the F1 mice testis is a consequence of interplay with the increased AhR. Nevertheless, upregulation of

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ER␣ would allow more estrogens to bind the receptor resulting in increased activation of ER␣ and estrogen responsiveness. Exposure of male rodents to estrogenic molecules elicits testicular dysfunctions including reductions in testis weight, level of testosterone, sperm count and motility (Goyal et al., 2001; Warita et al., 2006). Measurement of AGD is a common and sensitive index of reproductive toxicity in both humans and rodents (Arbuckle et al., 2008), and numerous studies have validated AGD as a sensitive marker for the effects of in utero exposure to endocrine disruptors (McIntyre et al., 2001; Wolf et al., 2004). In male rodents, perineal growth and caudal migration of the genital tubercle are dihydrotestosteronedependent (Bowman et al., 2003). Thus, AGD is considered to be a sensitive marker of prenatal androgen action. The neonate offspring of mice maternally exposed to B[b]F exhibited increased AGD and body weight (Table 1). The increased AGD mediated by the B[b]F exposure seems to suggest an androgenic property of B[b]F in the male offspring. However, we failed to detect any significant change in the serum androstenedione levels, steroidogenic enzyme expression, or testicular AR expression from young adult F1 offspring (Supplementary Fig. 1). The discrepancy may partly because either AGD is such a sensitive end-point or B[b]F-induced AGD lengthening is not due to its androgenic activity. At present, there are conflicting studies regarding the effect of PAHs on sex hormone receptors as some PAHs are suggested to be antiandrogenic, whereas some are estrogenic or antiestrogenic (Abdelrahim et al., 2006; Arcaro et al., 1999; Chaloupka et al., 1992; Kummer et al., 2008; Vinggaard et al., 2000). Therefore, more comprehensive in vivo animal studies for each congener and mixture of PAHs are needed because PAHs are mainly generated as part of larger mixtures, the compositions of which are different depending on the generation process, making it difficult to assess human toxicity and risk. Apoptosis is a homeostatic mechanism for maintaining the cell population throughout the development and aging of an organism. The Bcl-2 family of proteins is composed of subfamilies of prosurvival and pro-apoptotic members and is a critical component of diverse cellular apoptotic responses by regulating mitochondrial cell death activities (Dewson et al., 2008). BH3 domain-only proteins of the Bcl-2 family, also known as death ligands, include Bim, Bad, Bid, and Noxa, which are required for the activation of the Bak and Bax proteins through direct interaction (Jabbour et al., 2009). Bak and Bax are two death effector molecules that directly act at the mitochondrial outer membrane and are responsible for dismantling mitochondria through their oligomerization and the consequent release of cytochrome c into the cytosol (Chipuk et al., 2006). Increased apoptosis by different PAH congeners exposed in vitro has been reported previously (Raychoudhury and Kubinski, 2003; Staal et al., 2007), and PAH-induced expression of Bax was previously demonstrated in mouse oocytes (Matikainen et al., 2001). However, the activity of B[b]F on testicular apoptosis signaling molecules has not been known previously. In the current study, we found that the testes of F1 offspring maternally exposed to B[b]F exhibited more apoptotic cell death than control offspring (Fig. 4A). B[b]F exposure preferentially increased the expression of Bax as well as the level of Noxa, Bad, and Bim but did not affect Bak level (Fig. 4B–F). Therefore, the increased expression of Noxa, Bad, and Bim would be expected to further enhance Bax activation to disrupt the mitochondrial membrane, which may at least partly influence the poor sperm quality of the male offspring maternally exposed to B[b]F. Currently, very limited numbers of studies are available examining reproductive toxicities after perinatal exposure to PAHs. MacKenzie and Angevine showed that male mice exposed to 10, 40, and 160 mg/kg body weight benzo[a]pryene (B[a]P) in utero were infertile along with germ cell aplasia and had smaller seminiferous tubules compared to controls (MacKenzie and Angevine, 1981).

However, the exposure doses of B[a]P employed were very high considering the estimated mean dietary intake of PAHs for a standard male adult range between 6.72 and 12.04 ␮g/day (Martorell et al., 2010). Thus, in the current study, we chose to expose dams to a different type of PAH, B[b]F, at much lower doses, and their offspring started to show dysfunctions at 2 ␮g/kg body weight B[b]F. This dose of B[b]F may still higher than the concentration detected in human specimens, but the current mouse data may still can be useful for human risk assessment because humans are constantly exposed to a diverse array of PAHs and other toxicants for the period of lifetime. Recent epidemiology studies have demonstrated that increased urinary metabolites of PAHs are associated with decreased sperm quality and idiopathic infertility risks in Chinese males (Xia et al., 2009a,b). Therefore, the current transgenerational animal study implies that consumption of PAH-contaminated diets by mothers may possibly influence their offspring to cause dysfunctional male reproductive function in humans. Disclaimer None. Conflicts of interest Authors declare that there are no conflicts of interest. Acknowledgements This work was supported by the Priority Research Centers Program (2009-0093821) through the National Research Foundation of Korea (NRF) of the Ministry of Education, Science and Technology and by a grant (A084923) from the Korea Healthcare Technology R&D Project from the Ministry of Health, Welfare and Family Affairs. The authors are thankful to Jae-Hong Kim and Miae Won at CHA University for their helpful assistance regarding the in vivo animal experiment. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.toxlet.2011.03.003. References Abdelrahim, M., Ariazi, E., Kim, K., Khan, S., Barhoumi, R., Burghardt, R., Liu, S., Hill, D., Finnell, R., Wlodarczyk, B., Jordan, V.C., Safe, S., 2006. 3-Methylcholanthrene and other aryl hydrocarbon receptor agonists directly activate estrogen receptor alpha. Cancer Res. 66 (4), 2459–2467. Arbuckle, T.E., Hauser, R., Swan, S.H., Mao, C.S., Longnecker, M.P., Main, K.M., Whyatt, R.M., Mendola, P., Legrand, M., Rovet, J., Till, C., Wade, M., Jarrell, J., Matthews, S., Van Vliet, G., Bornehag, C.G., Mieusset, R., 2008. Meeting report: measuring endocrine-sensitive endpoints within the first years of life. Environ. Health Perspect. 116 (7), 948–951. Arcaro, K.F., Yang, Y., Gierthy, J.F., 1999. Benzo[k]fluoranthene enhancement and suppression of 17beta-estradiol catabolism in MCF-7 breast cancer cells. J. Toxicol. Environ. Health A 58 (7), 413–426. Bowman, C.J., Barlow, N.J., Turner, K.J., Wallace, D.G., Foster, P.M., 2003. Effects of in utero exposure to finasteride on androgen-dependent reproductive development in the male rat. Toxicol. Sci. 74 (2), 393–406. Carlsen, E., Giwercman, A., Keiding, N., Skakkebaek, N.E., 1992. Evidence for decreasing quality of semen during past 50 years. BMJ 305 (6854), 609–613. Carreau, S., Bilinska, B., Levallet, J., 1998. Male germ cells: a new source of estrogens in the mammalian testis. Ann. Endocrinol. (Paris) 59 (2), 79–92. Chaloupka, K., Krishnan, V., Safe, S., 1992. Polynuclear aromatic hydrocarbon carcinogens as antiestrogens in MCF-7 human breast cancer cells: role of the Ah receptor. Carcinogenesis 13 (12), 2233–2239. Chipuk, J.E., Bouchier-Hayes, L., Green, D.R., 2006. Mitochondrial outer membrane permeabilization during apoptosis: the innocent bystander scenario. Cell Death Differ. 3 (8), 1396–1402. Chung, J.Y., Kim, J.Y., Kim, Y.J., Jung, S.J., Park, J.E., Lee, S.G., Kim, J.T., Oh, S., Lee, C.J., Yoon, Y.D., Yoo, Y.H., Kim, J.M., 2007. Cellular defense mechanisms against

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