Perinatal exposure to 4-nonylphenol affects adipogenesis in first and second generation rats offspring

Toxicology Letters 225 (2014) 325–332

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Perinatal exposure to 4-nonylphenol affects adipogenesis in first and second generation rats offspring Hong-yu Zhang a,b,c,d , Wei-yan Xue a,b,c,d , Yuan-yuan Li a,b,c,d , Yue Ma a,b,c,d , Ying-shuang Zhu a,b,c,d , Wen-qian Huo a,b,c,d , Bing Xu a,b,c,d , Wei Xia a,b,c,d,∗ , Shun-qing Xu a,b,c,d,∗ a

Key Laboratory of Environment and Health, Ministry of Education, Wuhan 430030, China State Key Laboratory of Environment Health (Incubation), Wuhan 430030, China c Key Laboratory of Environment and Health (Wuhan), Ministry of Environmental Protection, Wuhan 430030, China d School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China b

h i g h l i g h t s • Perinatal exposure to 4-NP alters adipogenesis in the next two generations. • ER␣ signaling pathway was involved in the 4-NP’s effect.

a r t i c l e

i n f o

Article history: Received 21 October 2013 Received in revised form 12 December 2013 Accepted 13 December 2013 Available online 2 January 2014 Keywords: 4-NP Adipogenesis Two generation offspring

a b s t r a c t Maternal exposure to 4-nonylphenol (4-NP) during pregnancy was shown to alter adipogenesis in rodents, yet whether the effects are restricted to 4-NP-exposed offspring only or can be transmitted to the next generation are not known. Pregnant Wistar rats received either vehicle or 4-NP (5, 25 and 125 ␮g/kg/day) from gestation to postnatal day 21. F1 pups were subjected to blood biochemistry tests, or killed to obtain their gonadal fat to determine gene expression. Some F1 adult female rats were mated with F1 males from control group to obtain F2 pups, but without any exposure to 4-NP in the perinatal stage. F2 pups underwent studies similar to those performed on F1 pups. Serum total cholesterol, leptin levels were significantly elevated, the quantity and size of fat cells were increased, gene expression of key regulators of adipogenesis and lipogenic pathway of fat tissue were perturbed by 4-NP (p < 0.05 or p < 0.01). In addition, the expression of mRNA levels and protein of ER␣ were downregulated in adipose tissue in the two generation offspring. Perinatal exposure to 4-NP affects the adipogenesis in both male and female F1 offspring, and this effect can be progressed to the F2 offspring through the maternal line. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Over the past decades, the prevalence of obesity and associated metabolic diseases has risen steadily around the world, including the developed and undeveloped countries. Recent studies have suggested that higher incidence of obesity is correlated with the genetic factors, unhealthy life style, work stress and other factors. The increasing environmental endocrine disruptors (EDCs) are also important pathogenic factors (Holtcamp, 2012), such as bisphenol A (BPA), monoethylhexyl phthalate (MEHP) and 4-NP.

∗ Corresponding authors at: School of Public Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China. Tel.: +86 27 83657705; fax: +86 27 83657781. E-mail addresses: [email protected] (W. Xia), [email protected] (S.-q. Xu). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.12.011

4-NP is the final degradation product of alkylphenol polyethoxylates, which are widely used in many fields, such as textiles products, detergents, plasticizers, cosmetics, food packaging and other formulated products in our daily lives. Thus, 4-NP can pass into our body through many routes, such as drinking water, diet, skin contact, which will not cease during our lives. Under combination of the estrogen receptor ␣ (ER␣) (Shelby et al., 1996), EDCs will interfere with endocrine system and produce adverse effects to both human body and wildlife (Bechi et al., 2010; BonefeldJorgensen et al., 2007). Animal studies have demonstrated that: when 4-NP was exposed during the critical period of development, including fetal and/or early postnatal periods, it can induce permanent alterations in adipose tissue and then obesity (Chang et al., 2012; Hao et al., 2012). Large numbers of epidemiologic evidence have showed that developmental programming can be perpetuated to subsequent

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generations, even in the absence of further environmental stressors during intra-uterine and early postnatal life (Ding et al., 2012; Jimenez-Chillaron et al., 2009). For example, intra-uterine hyperglycemia impaired glucose tolerance and abnormal insulin levels in both F1 and F2 offspring (Ding et al., 2012). High dose of 4-NP (≈100–350 mg/kg/day), exposed in gestational period, produced consistent reproductive changes in three generations of pups (Chapin et al., 1999). One major hypothesis, to explain fetal programming of 4-NP exposure, is ER␣ signaling pathway. ER␣ is expressed in adipose tissue (Mizutani et al., 1994). The estrogen-like activity of 4-NP occurs through the expression of ER␣ silent gene (Vivacqua et al., 2003). When the ER␣ gene is deleted, the adiposity for both sexes will increase compared with their age-matched wild-type counterparts (Heine et al., 2000; Musatov et al., 2007). Epidemiological survey shows that ER␣ gene polymorphism has increased fat mass and increased waist–hip ratios (an index of intra-abdominal adiposity) relative to those with the normal genotype (Stevens-Lapsley and Kohrt, 2010). The additional expression of ER␣ silent gene can be passed on to next generation (Ohyama et al., 2007), and it will predispose to increased intra-abdominal adiposity and its related disease. However, how 4-NP programs the fatty in the subsequent generations has not been studied clearly yet. Despite the role of ER␣ in male and female WAT has been studied in many articles, it is unknown whether ER␣ normally plays any role in the intergenerational programming by 4-NP or not. Thus, we used an experimental rat model to check whether 4-NP can alter developmental programming of adipose tissue or not, and study further the potential mechanism involved by ER␣ signaling pathway. 2. Materials and methods 2.1. Maintenance and treatment of animals The animals were treated humanely with the minimum suffering during experiment, and all the procedures was approved by the Ethics Committee of Tongji Medical College (Huazhong University of Science and Technology, Wuhan, China). Virgin female and male genitor Wistar rats (8–10 weeks of age; from Vital Rivers, Peking, China) were housed in cages in a temperature-controlled room (22–25 ◦ C), a 12-h light/dark cycle and a relative humidity of 50 ± 10%. Polypropylene water bottles and polypropylene cages were used in this study. To avoid interfering experimental results by other environmental estrogens, phyto-oestrogens deficient diet (Shanghai Laboratory Animal Center, Shanghai, China; which contains 13.21% fat, 27.18% protein, and 59.61% carbohydrates, with energy of 14.39 kJ/g kcal/g), and ultra-pure water were provided ad libitum. The day of sperm-positive smears was considered to be gestation day (GD) 0. Pregnant rats were randomly divided into four groups that received olive oil as the control (Sigma–Aldrich, St. Louis, MO, USA; CAS no. 8001-25-0), 5 ␮g/kg/day of NP (NP-5), 25 ␮g/kg/day of NP (NP-25) and 125 ␮g/kg/day of NP (NP-125) from GD 6 till postnatal day (PND) by gavage. 4-NP were dissolved in olive oil, and dosage was adjusted daily for maternal body weight (0.5 ml/kg) every rat on GD 6 until the PND 21. After spontaneous delivery, the pups were adjusted to 6 (3 male and 3 female pups) per dam. The pups were maintained and designated as the first-generation offspring (F1). When weaned, 8

male and female pups were chosen randomly (one or two male/female pups per litter) to kill, and their trunk blood were collected and stored frozen at −80 ◦ C. The other pups were fed normal diet until 9 weeks old, and the composition of normal diet was adopted as Jie Wei’s article (Wei et al., 2011). At age of 9 weeks, 16 male offspring of F1 rats from control group were mated with control or experimental groups (8 female pups per groups) to produce the F2 offspring. The others were raised and sacrificed by decapitation until 26 weeks, trunk blood and adipose tissue were collected and stored frozen at −80 ◦ C. On PND 21 of F2 offspring, 8 male pups and 8 female pups were selected from each litter (one male/female pups per litter) to be maintained and given normal diet until the end of the experiment (Fig. 1). In this study, the concentration of 4-NP in diet and ultra-pure water were lower than the liquid chromatography tandem-mass spectrometry (LC–MS/MS)’s test line. Body weight was measured every week from the third week to the end of study. 2.2. Detection of 4-NP content Except the three types of diets and drinking water, the concentration of 4-NP in serum samples from the pups in F1 and F2 offspring on PND21 was analyzed. The procedures for sample pretreatment and the determination of 4-NP by API 3000 LCMS/MS were reported in previous reports (Guenther et al., 2002; Xiao et al., 2006). The detection limit for 4-NP was 0.5 ng/ml. 2.3. Biochemical assays After the animals were fasted overnight for 16 h, serum samples were analyzed for triglycerides (TG), blood-glucose (GLU) by automatic blood analyzer (Mindray BS-200; Shenzhen, China). Serum nonesterified free fatty acids (NEFA) were measured by colorimetric assay (Jiancheng Biological Institute, Nanjing, China). Leptin and adiponectin concentrations were determined by the ultrasensitive mouse insulin enzyme-linked immunosorbent assay (ELISA) (Linco Research, Millipore, Billerica, MA, USA). 2.4. Determination of adipocyte quantity and area Perigonadal white adipose dissected out from 26-week-old F1 rats and 13-weekold F2 rats were washed by phosphate buffer saline (PBS), weighted and calculated organ coefficients. Then, the white adipose tissue from gonadal fat pads were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 ␮m) were cut and stained with hematoxylin and eosin (H/E). Light microscope (Olympus CKX41, Japan) was used to evaluate for each specimen. The quantity and areas of 120 adipocytes were measured in captured images. Photographs were taken by using a Zeiss Axiocam Camera (Carl Zeiss, Gottingen, Germany) and assembled in Photoshop 6.0 (Adobe Systems, Mountain View, CA, USA). 2.5. Quantitative real-time PCR (RT-PCR) Total RNA was extracted from gonadal fat by using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized from total 2 ␮g RNA by RevertAid First Strand cDNA Synthesis Kit (Fermentas, China). The relative expression of mRNA was determined by RT-PCR using an ABI PRISM 7900 sequence detection system (Applied Biosystems, Foster City, CA, USA). RT-PCR was carried out in triplicates using Power SYBR Green PCR Master Mix reagents (Applied Biosystems, Framingham, MA, USA). Relative gene expression was calculated by the 2−Ct method with 36B4 as an endogenous reference gene. The primers are listed in Supplemental Table 1. 2.6. Western blot analysis Western blotting was performed as previously detailed (Meltser et al., 2008; Tiano et al., 2011). Antibodies for ER␣ (ab-2746; 1:1000) were obtained from Abcam

Fig. 1. Experimental design, including breeding scheme for F2 offspring.

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Table 1 Body weight, organ coefficient, serum leptin, adiponectin, TG, Glu and NEFA in F1 generation offspring. Control

NP-5

Male Body weight (g) Organ coefficient (%) Leptin (␮g/) Adiponectin (␮g/) TG (mmol/l) Glu (mmol/l) NEFA (␮g/ml)

384 2.32 4.52 111 0.68 7.83 644

Female ± ± ± ± ± ± ±

43 0.44 0.14 4 0.15 0.14 149

231 2.95 4.04 114 0.72 6.70 599

± ± ± ± ± ± ±

NP-25

Male 8 0.35 0.24 5 0.21 0.88 78

453 4.04 5.10 97 0.52 10.02 675

Female ± ± ± ± ± ± ±

51* 0. 26* 0.06* 3 0.09 0.23* 45

260 4.96 5.28 98 0.84 7.08 633

± ± ± ± ± ± ±

NP-125

Male 13** 0.15** 0.18* 6* 0.06 0.76 98

475 4.50 5.63 95 1.08 10.15 695

Female ± ± ± ± ± ± ±

57* 0.16** 0.80** 5* 0.22** 0.91* 24

260 5.52 5.56 95 1.08 9.99 676

± ± ± ± ± ± ±

Male 12** 0.69** 0.61** 7* 0.39* 0.43** 77

509 5.56 6.73 84 1.47 10.22 701

Female ± ± ± ± ± ± ±

29** 1.05** 0.09** 3** 0.22** 0.16* 133

284 6.78 5.97 89 0.83 10.44 701

± ± ± ± ± ± ±

3** 0.42** 0.49** 5* 0.13 0.41** 179

Data are means ± SD. Significance was determined by ANOVA in female F1 dams (n = 8). * p < 0.05 vs. control. ** p < 0.01 vs. control.

plc (Cambridge, USA). ␤-Actin (sc-47778; 1:500) and goat anti-mouse IgG-HRP (sc2005; 1:5000) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).

2.7. Statistical analysis All data were expressed as the means ± SE. Data were treated for statistical analysis using SPSS 13.0 software (SPSS Inc., Chicago, IL, USA). The different times of body weight were analyzed by repeated measures analysis of variance (ANOVA). Comparison between groups was made using one-way ANOVA followed by least significant difference (LSD) and Dunnett’s t-test. Values of p < 0.05 were considered to be statistically significant.

3. Results 3.1. 4-NP exposure increases first- and second-generation offspring body weight The mean 4-NP content in weaned rat blood from F1 NP-25 group and F1 NP-125 group was 2.48 ± 0.32 ng/ml and 13.13 ± 1.27,

respectively. In other groups, 4-NP was found below the detection of limit. 4-NP exposure did not change the gestation days, litter sizes, sex ratios and birth weights of F1 offspring (Supplementary Table 2). After fostering by normal diet, perinatal exposure to 4-NP significantly increased the body weight and adipose organ coefficient of F1 offspring compared with controls from 12 weeks till 26 weeks of age, which showed a dose–response relationship among the control group and treated groups at 26 or 13 weeks of age (Table 1 and Fig. 2A and B), though the average daily energy intakes were not different in the groups. To evaluate whether differences in body weight are related to alter the body weights of F0 generation, we measured the body weight of F0 pregnant rats from GD 6 to the end of gestation. But there were also not difference when compared with the control group. In F2 generation, the birth weight of male or female pups had more weight gain in the 4-NP groups than in the control groups (Supplementary Table 3). After weaned, we observed the average

Fig. 2. Effect of 4-NP on body weight of F1 generation (male: A; female: B; 26 weeks old) and F2 generation (male: C; female: D; 13 weeks old) rats. n = 7–8 animals per group. The marks from the lowest to the highest represent NP-5, NP-25 and NP-125 results respectively. × p > 0.05 vs. control; *p < 0.05 vs. control.;  p < 0.01 vs. control.

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Table 2 Body weight, organ coefficient, serum leptin, adiponectin, TG, Glu and NEFA in F2 generation offspring. Control

NP-5

Male Body weight (g) Organ coefficient (%) Leptin (␮g/) Adiponectin (␮g/) TG (mmol/l) Glu (mmol/l) NEFA (␮g/ml)

257 2.31 4.21 138 0.72 3.57 654

Female ± ± ± ± ± ± ±

25 0.64 0.47 7 0.21 0.77 132

205 2.31 4.74 125 0.73 3.34 602

± ± ± ± ± ± ±

NP-25

Male 7 0.40 0.52 6 0.18 0.74 76

322 4.24 4.84 93 0.78 4.66 679

Female ± ± ± ± ± ± ±

28* 0.26* 0.46* 3* 0.11 0.82* 150

223 3.06 5.43 103 0.77 4.44 652

± ± ± ± ± ± ±

NP-125

Male 11** 0.58 0.54* 5* 0.08 0.77* 123

332 4.70 4.95 81 1.15 4.91 732

Female ± ± ± ± ± ± ±

17** 0.16** 0.52* 2** 0.42* 0.78** 98

229 4.55 5.87 100 1.11 4.49 679

± ± ± ± ± ± ±

Male 15** 0.13* 0.62* 5** 0.42* 0.34* 44

330 5.76 5.09 74 1.42 5.08 743

Female ± ± ± ± ± ± ±

29** 0.94** 0.54** 6** 0.39** 1.39** 146

230 4.87 5.94 90 1.14 4.85 709

± ± ± ± ± ± ±

13** 0.37** 0.92** 6** 0.40* 0.93** 128

Data are means ± SE. Significance was determined by ANOVA in female F1 dams (n = 8). * p < 0.05 vs. control. ** p < 0.01 vs. control.

daily energy intakes were not different in 4-NP exposure groups and the control group. The body weight and organ coefficient of adipose tissue of 4-NP-exposed F2 offspring were also increased in a dose-dependent way compared with controls, indicating that weight gain was due to the accumulation of adipose tissue (Table 2 and Fig. 2C and D).

simple 4-NP groups, but not statistically significant in neither firstnor second-generation offspring.

3.2. 4-NP exposure induces biochemical abnormalities in the blood of first- and second-generation offspring (Tables 1 and 2)

To better characterize the increase in adipose tissue deposition observed in 4-NP-exposed offspring, we performed histological sections of their perigonadal adipose tissue. As shown in representative photographs of these tissues, adipocytes of F1 offspring (Fig. 3A) and F2 offspring (Fig. 3C) exposed to 4-NP appeared to be hypertrophied compared with those of control rats at 26 or 13 weeks of age. Moreover, perinatal exposure to 4-NP significantly increased the average area of adipocytes of both F1 and F2 offspring in a dose-dependent way (Fig. 3B and D) (p < 0.01).

Compared with the corresponding control groups, serum leptin, adiponectin and Glu in most of the 4-NP-exposed groups were significantly higher (p < 0.05 or p < 0.01), particular, the NP-125 (p < 0.01) in two generations. Outside of F1 female generation, TG had increased in two generations of rats and had statistically significant (p < 0.05 or p < 0.01). NEFA had increased gradually in the

3.3. 4-NP exposure increases the quantity and areas of adipocytes in first- and second-generation offspring

Fig. 3. Effect of perinatal exposure to 4-NP on average area sizes of epididymal and uterine fat in F1 and F2 male and female offspring (26 weeks and 13 weeks, respectively; n = 6–8 rats/group). (A) Representative histologic sections of epididymal and uterine fat tissue form control and 4-NP-exposed F1 offspring. (B) Average areas of fat in F1 offspring. (C) Representative histologic sections of epididymal and uterine fat tissue form control and 4-NP-exposed F2 offspring. (D) Average areas of fat in F2 offspring. Results are expressed as means ± SE. **p < 0.01 vs. control (ANOVA).

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3.4. 4-NP exposure changes mRNA expression of genes involved in adipogenesis and lipogenesis in adipose tissue of F1 and F2 offspring rats

lipoprotein lipase (Lpl) and fatty acid synthetase (Fas) were upregulated in perigonadal adipose tissue of 4-NP-exposed F1 and F2 offspring (p < 0.05) (Fig. 4A and B).

RT-PCR results showed that mRNA levels of the proadipogenic transcription factors sterol regulatory element-binding protein 1 (Srebp-1) and peroxisome proliferator-activated receptor alpha (Ppar-) were significantly increased in perigonadal adipose tissue of 4-NP-exposed F1 and F2 offspring (p < 0.05) (Fig. 4A and B). In addition, mRNA expression levels of lipogenic enzymes such as

3.5. 4-NP exposure decreases ER˛ expression in adipose tissue of first- and second-generation offspring rats The mRNA expression levels of ER␣ were reduced significantly in the perigonadal adipose tissue of 26-week-old F1 offspring compared with control rats (Fig. 5A). In the F2 offspring, the mRNA

Fig. 4. Relative mRNA levels of Lpl, Fas, Srebp-1 and Ppar- [in arbitrary units (AU)] in epididymal and uterine fat from males (A) and female (B) (n = 6–8 rats/group). Results are expressed as means ± SE, *p < 0.05 vs. control; **p < 0.01 vs. control.

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Fig. 5. The mRNA and protein expression levels of ER␣ in adipose tissue from F1 and F2 control and 4-NP exposed offspring (? weeks). (A) The mRNA expression levels of ER␣ in gonadal fat from F1 offspring. (B) The mRNA expression levels of ER␣ in gonadal fat from F2 offspring. (C) Western blot analysis of ER␣ protein expression in gonadal fat from F1 offspring. A representative photograph of western blot is shown. (D) Western blot analysis of ER␣ protein expression in gonadal fat from F2 offspring. A representative photograph of western blot is shown (n = 6–8 rats/group). Results are expressed as means ± SE. *p < 0.05 vs. control, **p < 0.01 vs. control.

expression levels of ER␣ were also significantly down-regulated in the perigonadal adipose tissue of 4-NP groups compared with control group (p < 0.05) (Fig. 4B). Western blot analysis showed that the protein levels of ER␣ (Fig. 4C and D) were consistent with the changes in mRNA levels. 4. Discussion In the present study, we observed significant effects of 4-NP exposure during pregnancy and lactation on the development of adipose tissue in the next two generations, affecting both the male and female offspring. The effects were accompanied by disturbed lipid homeostasis, increased gene expression involved with adipogenesis and lipogenesis in adipose tissue and decreased ER␣ expression. Previous studies have reported that exposure to high dose of 4NP (≈100–350 mg/kg/day) in gestational period induced consistent reproductive changes in three generations of pups (Chapin et al., 1999). In our study, the lowest dose used was only 5 ␮g/kg/day which is considered as an environmentally relevant concentration by the Danish Institute of Safety & Toxicity (Nielsen et al., 2000). 4-NP content was only found in the serum of NP-25 and NP-125

F1-generation offspring, but it was very low (under detection limit) in the serum of other groups. To mimic the most likely route of human exposure, 4-NP was gavaged from GD 6 to the end of lactation (PND21). This exposure window excluded the effects of 4-NP on the formation of eggs and sperm (Smallwood et al., 2011), but included the period that the majority of the genome was reprogrammed in the adipose tissue (Cristancho and Lazar, 2011). With the increase rate of obesity nowadays, numerous of studies point out environmental insults in pregnancy can induce longterm effects to the offspring, resulting in to obesity or other chromic adult disease (Cupul-Uicab et al., 2012; Snijder et al., 2013). And more importantly, some researchers have demonstrated that the similar metabolic phenotypes of the first-generation, associated with exposure to maternal abnormal condition, can also be observed in the second-generation offspring (Jimenez-Chillaron et al., 2009). For example, Clapp et al. (2002) demonstrated that infants of mothers on high-glycemic diets had higher birth weight and skinfold thickness than those exposed to a low-glycemic diets. At first, we observed that perinatal exposure to 4-NP increased the body weight, progenitor white adipose weight and size in the F1 offspring. The results are consistent with the findings reported by other studies (Chang et al., 2012; Hao et al., 2012).

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There is no significant difference in food intake in 4-NP exposure groups and the control group, so the enhanced weight gain was not the result of the increase in food intake. Also, we illustrate for the first time that exposure to 4-NP during the perinatal period of F0 generation changed adipogenesis and increased the adipose storage of F1 generation, and this phenotype can be passed on to the female and male F2 generation through maternal line. Moreover, the change in size of adipocytes in 4-NP-exposed offspring in F1 and F2 generation is associated with an overexpression of genes involved in adipogenesis and lipogenesis in adipose tissue, such as Ppar-, Srebp-1, Lpl and Fas. These observations are in consistent with the results induced by other known environmental obesogen in previous reports (Somm et al., 2009). Ppar- belongs to the nuclear receptor superfamily of ligand-dependent transcription factors, and Srebp-1 is a nuclear transcription factor. Both of them are proadipogenic transcription factors, which can stimulate the differentiation of preadipocytes into mature adipocytes and increase the quantity of fat cells (Kajita et al., 2012). The in vivo observations are in accordance with previous in vitro results, as 4-NP can accelerate the differentiation of 3T3-L1 fibroblasts into adipocytes (Hao et al., 2012; Masuno et al., 2003). Also, Ppar- can adjust the level of transcription of the metabolism of fatty acids (Zhang et al., 1996). Srebp-1, involving in the regulation of unsaturated fatty acids, TC and TG’s synthesis, plays an important role in regulation of lipid accumulation and the pathogenesis of obesity (Im et al., 2011). The gene expression of Lpl and Fas, key enzymes regulating lipid metabolism and fat cell size, was up-regulated in adipose tissue of F1 and F2 offspring exposed by 4-NP, corroborating the increased levels of the two enzymes in adipose and liver of rats offspring exposed with 4-NP in utero (Chang et al., 2012; Hao et al., 2012). Lpl can break down plasma triglycerides into NEFA and glycerol. Obese subjects have higher levels of Lpl activity in their adipose tissue compared to lean subjects (Pykalisto et al., 1975). Fas overexpression was linked with visceral fat accumulation (Berndt et al., 2007) and energy imbalance (Loftus et al., 2000). In addition, Fas is involved in Ppar- activation and adipogenesis in mouse embryonic fibroblasts (Lodhi et al., 2012). In our research, these findings provide the first evidence that 4-NP affects the gene expression related to the hyperplasia and differentiation of adipocyte in F1 and F2 offspring, which is correlated with abnormal adipose storage. As we all know, estrogens are known to be involved in the modulation of energy balance and body composition (Musatov et al., 2007). Adipocytes can produce estrogens via aromatization from androgenic precursors, and the estrogens content was consisted with the total body adiposity (Gavin et al., 2013). ER␣ is expressed in adipose tissue, and adjusts the proliferation and differentiation of adipose cell (Pedersen et al., 2004). When the ER␣ gene is deleted in mice, the adiposity of both sexes increases compared with their age-matched wild-type counterparts (Heine et al., 2000). These data indicate that ER␣ signaling pathway is critical for regulating WAT deposition. 4-NP reduced the expressions of ER␣ in adipose tissue of F1- and F2-offspring in a dose-dependent way in our work, accompanied with the remarkably increasing of adiposity. 4-NP can bind to the ER␣ but do not activate transcription (Adachi et al., 2005; Shelby et al., 1996). Hence, the down-regulation of ER␣ will increase fat cell proliferation and differentiation. So, the expression of genes involved in lipogenesis, for instance, Lpl, Fas, Srebp-1 and Ppar-, increased significantly in comparison with the corresponding control groups (Iverius and Brunzell, 1988; Liu et al., 2008). Besides, epigenetic modifications are thought to correlated with the transgenerational effects induced by environmental factors (Anway et al., 2005, 2006), it is possible that 4-NP may change the epigenetic modifications which needs further study.

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