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Effects of Nonylphenol on Brain Gene Expression Profiles in F1 Generation Rats1
BIOMEDICAL AND ENVIRONMENTAL SCIENCES 21, 1-6 (2008)
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Effects of Nonylphenol on Brain Gene Expression Profiles in F1 Generation Rats1 YIN-YIN XIA, PING ZHAN2, AND YANG WANG Department of Environment Hygiene, Chongqing University of Medical Sciences, Chongqing 400016, China
Objective To explore the effects of nonylphenol on brain gene expression profiles in F1 generation rats by microarray technique. Methods mRNA was extracted from the brain of 2-day old F1 generation male rats whose F0 female generation was either exposed to nonylphenol or free from nonylphenol exposure, and then it was reversely transcribed to cDNA labeled with cy5 and cy3 fluorescence. Subsequently, cDNA probes were hybridized to two BiostarR-40S cDNA gene chips and fluorescent signals of cy5 and cy3 were scanned and analyzed. Results Two genes were differentially down-regulated. Conclusion Nonylphenol may disturb the neuroendocrine function of male rats when administered perinatally. Key words: Nonylphenol; cDNA microarray; Brain tissue
the effects of NP on the early brain development of F1 male rats by cDNA microarray, and to testify the hypothesis that NP has the potential to adversely affect hormonal status.
INTRODUCTION Nonylphenol (NP) is generated from alkylphenol ethoxylates that are widely used in the production of plastics, textiles as detergents, paints, pesticides, and cosmetics[1]. NP has adverse effects on the development of reproductive tract of the male animals which were exposed to it perinatally[2], including reduced size of testes, decreased sperm production, cryptorchidism, and reduced reproductive organ weights. On the other hand, no effects of NP on male reproduction have been found[3]. It was also reported that NP possesses the estrogenic property in vitro and in vivo systems[4]. Brain is the source of behavior, thoughts, feelings, and experiences. If a rodent with functional male genitals is deprived of androgens immediately after birth, its male sexual behavior will be reduced, and more female sexual behaviors will take place instead. These lifelong effects of early exposure to sex hormones are characterized by ‘organizational’ changes because they appear to alter brain function permanently during a critical period in prenatal or early postnatal development. Administering the same sex hormones at later stages or in adults has no such similar effects[5]. The aim of the present study was to investigate
MATERIALS AND METHODS Animals Ten adult male Sprague-Dawley rats (300-350 g) and twenty adult female Sprague-Dawley rats (230-260 g) were purchased from Laboratory Animal Center (West China Center of Medical Sciences), Sichuan University. The animals were housed in a temperature-controlled room with free access to under running water in a 12 h/ dark and light cycle. The F0 rats were housed for 1 week prior to mating. Female rats and male F0 rats were mated with 2:1 proportion overnight and the day when copulatory plugs were found was designated as GD-0. The GD-0 rats were randomly assigned to either treatment or control group. Rats in treatment group were daily given 200 mg/kg NP by oral gavage from GD 7 to PND 2 (day of birth=postnatal day 0), while those in control group received an equivalent volume of corn oil. The filial rats procreated from pregnant F0 rats were denominated first generation rats (F1).
1
This research was supported by the Innovation Fund of Chongqing University of Medical Sciences (No. 200417). Correspondence should be addressed to Ping ZHAN. Tel: 86-23-68485803. Fax: 86-23-68485008. E-mail: [email protected] Biographical note of the first author: Yin-Yin XIA, M. D, female, born in 1978, majoring in endocrine disrupting chemicals. 2
0895-3988/2008 CN 11-2816/Q Copyright © 2008 by China CDC 1
2
XIA, ZHAN, AND WANG
Tissue Samples Brain tissue samples were obtained from PND-2 F1 rats, quickly frozen in liquid nitrogen for RNA extraction. Chip Preparation Four thousand and ninety-six target cDNA clones were used in the rat cDNA chip (provided by Biostar Genechip Inc., Shanghai, China). These genes were amplified by PCR using universal primers and purified with standard method. The quality of PCR was monitored by agarose electrophoresis. Target genes were dissolved in 3×SSC spotting solution, and spotted on silylated slides (Telechem, Inc., USA) by Cartesian 7500 spotting robotics (Cartesian, Inc., USA). Each target gene was dotted twice. After spotting, the slides were hydrated (2 h), dried (0.5 h, room temperature), cross-linked under UV light and treated with 0.2% SDS, H2O and 0.2% NaNBH4 for 10 minutes, respectively. The slides were then dried again for use. Probe Preparation Total RNA in NP-treated and control F1 rat brain tissues was extracted as previously described[6]. The tissues were removed from liquid nitrogen and ground into tiny powder in a ceramic mortar, to which liquid nitrogen was added, and homogenized in D-solution plus 1% mercaptoethanol. After centrifugation, the supernatant was extracted twice with phenol: chloroform (1:1), then once with NaAC and acidic phenol: chloroform (5:1). The aqueous phase was precipitated by equal volumes of isopropanol. The precipitate was centrifuged and dissolved with Millie-Q H2O. After purification by a LiCl precipitating method, UV analysis and electrophoresis detection showed the good quality of purified RNA. mRNA was isolated and purified using an Oligotex mRNA Midi kit (QIAGEN, Inc. USA). The fluorescein-labeled cDNA probe was prepared through retro-transcription and purified, using the method of Schena. cDNA probes from brain tissue of control F1 rats were specifically labelled with Cy3-dUTP and those from brain tissue of NP-treated F1 rats were labelled with with Cy5-dUTP. The probes were mixed (Cy3-dUTP control + Cy5-dUTP ZP-1, Cy3-dUTP control + Cy5-dUTP ZP-2), precipitated by ethanol, and resolved in 20 μL hybridization solution (5×SSC+0.2% SDS). Hybridization and Washing Hybridizing probe and two same chips were denatured in a 95℃ bath for 5 minutes. The probe
was added onto the chips and covered with glass. The chips were hybridized in a sealed chamber at 60℃ for 15-17 h. After the cover glasses were removed, the slides were washed in a solution of 2×SSC+0.2% SDS, 0.1×SSC+0.2% SDS and 0.1% SSC, 10 minutes each, respectively, then dried at room temperature. Fluorescence Scanning and Analysis The chips were scanned with a Scan Array 3000 scanner (General Scanning Inc., USA). The overall intensity of Cy3 and Cy5 was normalized and corrected by a coefficient according to the location ratios of the 40 housekeeping genes. The acquired image was analyzed by ImeGene 3.0 software. Intensity of the fluorescent signal and each ratio of Cy3 to Cy5 were compared. The data were obtained on an average of two repeated spots. The criteria for screening the differentially expressed genes were defined as follows. The absolute value of the natural logarithm of the signal ration of Cy5/Cy3 was greater than 0.69 (gene expression change >2 folds), one of the signal values of Cy3 and Cy5 was greater than 600, and the results of PCR were good. RESULTS The fluorescent scanning profile of gene expression is shown in Fig. 1. Cy3-labeled cDNA probes from normal F1 rat brain tissues and Cy5-labeled cDNA probes from NP-treated F1 rat brain tissues, ZP-1 and ZP-2, were hybridized through microarray. The red spot indicated the upregulated genes, the green spot indicated the down-regulated genes, and the yellow spots indicated genes which had similar expression between the treated F1 rat brain tissues and normal tissues. The scatter plots, plotted from Cy3 and Cy5 fluorescent signal values, revealed a dispersed distribution pattern. Most spots were found along an almost 45° diagonal line. Red spots showed signal differences between 0.5 to 2.0 folds. Certain yellow spots were distributed away from the 45° diagonal line, indicating the existence of many differentially expressed genes. The difference in these signals between control and NP-treated groups was greater than 2.0 folds or less than 0.5 folds (beyond the range of 0.5-2.0 folds) (Fig. 2). Since red indicates the signal difference between 0.5 to 2.0 folds, while yellow represents the signal difference beyond the range of 0.5-2.0 folds, these yellow spots located away from the diagonal line indicated the existence of several differentially expressed genes caused by NP treatment, as shown in Fig. 2.
EFFECTS OF NONYLPHENOL ON BRAIN GENE EXPRESSION
3
FIG. 1. Overlay of hybridizing signals labelled by two-color fluorescence on the gene chip ZP-1 (left), and ZP-2 (right).
FIG. 2. Scatter plots of gene expression pattern, ZP-1(left), and ZP-2 (right).
Seven genes were differentially expressed on ZP-1 gene chip, indicating the altered gene expression caused by NP treatment. The expression of all these genes was down-regulated by 2 folds (ratio<0.5) (Table 1). Among these 7 genes, 2 encoded proteins involved in metabolism of substance and energy, namely stearyl-CoA desaturase (SCD) and glutathione
s-transferase M5. Another 4 genes encoded proteins involved in cell structures and communications, namely endosulfine alpha (Ensa), protein tyrosine phosphatase, receptor type, W (Esp), protein tyrosine phosphatase 2E (PTP2E), actin-related protein 3 (Arp3). Cyp8b1 gene was shown to be a member of cytochrome P450 superfamily.
TABLE 1 List of Differential Expressed Genes, ZP-1 Genbank_ID
Gene Name
Ratio
J02585
Stearyl-CoA Desaturase (SCD)
0.321
U86635
Glutathione s-transferase M5
0.415
Endosulfine alpha (Ensa)
0.420
NM_033099
Protein Tyrosine Phosphatase, Receptor Type, W (Esp)
0.451
NM_031241
Cytochrome P450, 8b1, Sterol 12 alpha-hydrolase (Cyp8b1)
0.488
Protein Tyrosine Phosphatase 2E (PTP2E)
0.490
Actin-related Protein 3 (Arp3)
0.492
NM_021842
U17971 AF307852
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XIA, ZHAN, AND WANG
Sixteen differentially expressed genes were detected on ZP-2 gene chip. Among them, 3 genes were up-regulated by>2.0 folds (ratio>2), while 13 genes were down-regulated by >2.0 folds (ratio<0.5), as indicated in Table 2. Among the 16 genes, 5 encoded proteins involved in metabolism of substance and energy, namely stearyl-CoA desaturase (SCD), liver glycogen phosphorylase (Pygl), peroxisomal 2,4-dienoyl CoA reductase (px-2,4-DCR), succinate dehydrogenase complex, subunit A (SDHA), gonadotropin-regulated long chain acyl-CoA synthetase
(GR-LACS). Another 6 genes encoded proteins involved in cell structures and communications, namely zonula occludens 2 protein (ZO-2), endosulfine alpha (Ensa), ERM-binding phosphoprotein (LOC59114), glutamate receptor (AMPA2), integral membrane-associated protein 1 (Itmap1), transglutaminase 1 (Tgm1). Three genes were found to be members of cytochrome P450 superfamily, namely CYP3A3, CYP4B1, and CYP2C23. The rest were found to be some unsorted genes namely E5E antigen, sarcomeric muscle protein.
TABLE 2 List of Differentially Expressed Genes, ZP-2 Genbank_ID
Gene Name
J02585 NM_013105
Ratio
Stearyl-CoA Desaturase (SCD)
0.236
Cytochrome P450, Subfamily IIIA, Polypeptide 3 (Cyp3a3)
0.247
U75916 NM_022268 NM_021842 AF021854
Zonula Occludens 2 Protein (ZO-2)
0.317
Liver glycogen Phosphorylase (Pygl)
0.330
Endosulfine alpha (Ensa)
0.403
Peroxisomal 2,4-dienoyl CoA Reductase px-2,4-DCR#1
0.443
D37934
5E5 Antigen
0.450
NM_021594
ERM-binding Phosphoprotein (LOC59114)
0.452
NM_017261
Glutamate Receptor, Ionotropic, AMPA2 (alpha 2) (Gria2)
0.454
NM_054005
Integral Membrane-associated Protein 1 (Itmap1)
0.461
AB072907
SDHA mRNA for Flavoprotein Subunit of Succinate-ubiquinone Reductase
0.479
AF208125
Gonadotropin-regulated Long Chain Acyl-CoA Synthetase (GR-LACS)
0.484
NM_031659
Transglutaminase 1, K Polypeptide (Tgm1)
0.485
NM_016999
Cytochrome P450, Subfamily IVB, Polypeptide 1 (Cyp4b1)
2.029
NM_031839
Arachidonic Acid Epoxygenase (Cyp2c23)
2.258
NM_057191
Sarcomeric Muscle Protein (Sarcosin)
2.700
When exposed to NP, statistically significant differential expression was found in 2 genes: ZP-1 and ZP-2. These 2 genes were both suppressed (ratio <0.5), the immediate toxic response appeared to be mediated by the transcriptional regulation of the common genes (Table 3). Two genes, SCD and Ensa, were found to be
differentially expressed simultaneously on both ZP-1 and ZP-2 chips, as shown in Table 3. Both of them were down-regulated (ratio<0.5), indicating that immediate toxic response after nonylphenol exposure could be mediated by the transcriptional regulation of common genes.
TABLE 3 List of Differentially Expressed Genes, ZP-1 and ZP-2 Genbank_ID J02585
Gene Name
Ratio (ZP-1)
Ratio (ZP-2)
Stearyl-CoA Desaturase (SCD)
0.321
0.236
Endosulfine alpha (Ensa)
0.420
0.403
NM_021842
DISCUSSION The study was to characterize the immediate biological responses of F1 male rat brain tissue to
toxic dose of NP and to use this information to develop biomarkers for its toxicity[7]. We used DNA microarray technology to detect NP-induced alterations in gene expression during early brain
EFFECTS OF NONYLPHENOL ON BRAIN GENE EXPRESSION
development of F1 generation male rats. These 23 genes were differentially expressed, involving 7 genes of ZP-1 gene chip and 16 genes of ZP-2 gene chip, representing a broad range of mRNAs encoding diversified functionalities, including metabolism of substance and energy, cell structures, and communications, such as receptors and signaling molecules that regulate the electrophysiological properties of neurons, action maintaining the structural integrity of neurons, as factors regulating fat and protein degradation. Among these 23 genes, however, only 2 were differentially expressed simultaneously on both ZP-1 and ZP-2 chips: Stearyl-CoA desaturase (SCD) gene, endosulfine alpha (Ensa). SCD is consisted of 358 amino acids, of which 62% are hydrophobic amino acids, corresponding to a molecular mass of 41 400 daltons[8]. SCD is a rate limiting enzyme in the biosynthesis of monounsaturated fats and is expressed in the liver, kidneys, brain, and adipose tissue. Oleic acid, a principal product of SCD, is the major unsaturated fatty acid in lipid stores of human adipose tissue and phospholipids in red blood cell membrane. The ratio of stearic acid to oleic acid has been implicated in the regulation of cell growth and differentiation through effects on cell-membrane fluidity and signal transduction[9]. In this study, the down-regulated SCD gene expression in brain tissue of NF-exposed PND-2 F1 rats suggested that NP could affect neuron growth and differentiation by interfering with the biosynthesis of unsaturated fatty acids. Leptin is recognized as the central mediator in an endocrine circuit-regulated energy homeostasis. Cohen et al.[10] found that SCD is specifically repressed during leptin-mediated weight loss, and that adiposity is significantly reduced in mice lacking SCD. These findings indicate that SCD plays an important role in some aspects of metabolic syndrome, suggesting that NP may be associated with energy homeostasis indirectly and a risk factor for metabolic syndrome. Ensa, first isolated from ovine brain as a possible endogenous ligand for sulfonylurea receptors, is expressed in brain, muscle, and endocrine tissues. It is a 121-amino acid-protein with a total molecular mass of 19 kDa, as well as a member of a highly conserved cAMP-regulated phosphoprotein (ARPP) family[11]. The sulphonylurea receptor is physically associated with a pore-forming subunit (Kir 6.2) to constitute the ATP-dependent potassium channel (KATP)[12]. By binding to SUR, Ensa induces closure of KATP channels on the pancreatic β-cells, leading to membrane depolarization, which, in turn, activates
5
voltage-gated L-type Ca2+-channels. The resulting calcium influx triggers insulin release[13]. Ensa is able to block KATP channels not only on the pancreatic β-cells, but also on brain, while the neurobiological role of alpha-endosulfine has not been studied yet. Kim and Lubec[14] examined the expression levels of Ensa in frontal cortex and cerebellum from patients with Down syndrome (DS), and found that considerably decreased Ensa could result in the continuous opening of KATP channels and the subsequent decrease of neurotransmitter release associated with cognition. Dou et al.[15] described that the majority of Ensa genes are expressed in pyramidal neurons of rat brain, which represents the principal excitatory neurons in various brain regions, and swimming-related stress causes persistent up-regulation of Ensa gene expression in several brain regions. In the present study, the down-regulated expression of Ensa gene in brain tissue of NP-exposed PND-2 F1 rats suggests that NP affects the level of insulin by decreasing the expression of Ensa. The decreased Ensa expression results in the continuous opening of KATP channels in brain, inhibits the depolarization of the cell membrane and the calcium influx, and regulates the release of neurotransmitters, such as acetylcholine. Consequently, it is likely that NP disturbs the release of pituitary hormones, such as gonadotropin releasing hormone. This is one of the probable mechanisms that NP impairs neuroendocrine functions by disrupting the synthesis and metabolism of steroid hormone. The response of organs to exposure of NP is often a combined response of various cell types, tissues, and organs within an individual. As brain plays an important role in regulating the metabolism and systemic function, proteins in brain tissue encoded by a specific gene can regulate functions of multiple systems, and proteins encoded by multiple genes regulate the functions of a specific system[16]. Fitch et al. reported[17] that mammalian sexual differentiation is primarily mediated by testicular androgens, and that exposure to androgens in early life leads to a male brain as defined by neuroanatomy and behavior. For F1 male rats, the sensitive period of sexual differentiation extends at least through the perinatal period (roughly from embryonic day 17 through postnatal days 8-10 in rodents). It is likely that exposure to NP during this period may disrupt homeostasis of steroid hormones, influence brain development and sexual differentiation of male rats. In conclusion, nonylphenol may disturb the neuroendocrine function of male rats when administered perinatally. The gene expression
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XIA, ZHAN, AND WANG
profiles derived from the present study provide insights into potential biomarkers of perinatal NP exposure and possible mechanisms of NP in disrupting neuroendocrine functions. ACKNOWLEDGEMENTS We thank Dr. Hao ZHANG, Department of Environment Hygiene, West China Center of Medical Sciences, Sichuan University, for providing nonylphenol and invaluable information, and Dr. Yuan-Feng LI, Tongji Medical College of Huazhong University of Science and Technology, for the strong support in data collection and manuscript preparation. REFERENCES 1. Naylor C G, Mierure J P, Weeks J, et al. (1992). Alkylphehol ethoxylates in the environment. J Am Oil Chemists 69, 695-703. 2. Boockfor F R, Blake C A (1997). Chronic administration of 4-tertoctylphenol to adult male rats causes shrinkage of the testes and male accessory sex organs, disrupts spermatogenesis, and increases the incidence of sperm deformities. Biol Reprod 57, 267-277. 3. Chapin R E, Delaney J, Wang Y, et al. (1999). The effects of 4-nonylphenol in rats: A multigenerational reproduction study. Toxicol Sci 52, 80-91. 4. Jobling M A, Samara V, Pandya A, et al. (1996). Recurrent duplication and deletion polymorphisms on the long arm of the Y chromosome in normal males. Hum Mol Genet 5, 1767-1775. 5. Kimura D (1992). Sex differences in the brain. Sci Am 267(3), 118-125.
6. Peng X L, Yuan H Y, Xie Y, et al. (1998). Experimental technique of gene engineering. HuNan Science and Technique Issue Agency, Changsha, pp. 197-199. 7. Kaiser S, Nisenbaum L K (2003). Evaluation of common gene expression patterns in the rat nervous system. Physiol Genomics 16(1), 1-7. 8. Thiede M A, Ozols J, Strittmatter P (1986). Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase. J Biol Chem 261(28), 13230-13235. 9. Lin Z, Lan G E, Satish P, et al. (1999). Human stearoyl-CoA desaturase : alternative transcripts generated from a single gene by usage of tandem polyadenylation sites. Biochem J 340, 255-264. 10. Cohen P, Ntambi J M, Friedman J M (2003). Stearoyl-CoA desaturase-1 and the metabolic syndrome. Curr Drug Targets Immune Endocr Metabol Disord 3(4), 271-280. 11. Virsolvy A, Smith P, Bertrand G, et al. (2002). Block of Ca(2+)-channels by alpha-endosulphine inhibits insulin release. Br J Pharmacol 135(7), 1810-1818. 12. Inagaki N, Gonoi T, Clement JP 4th, et al. (1995). Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270(5239), 1166-1170. 13. Ashcroft S J, Ashcroft F M (1990). Properties and functions of ATP-sensitive K-channels. Cell Signal 2(3), 197-214 14. Kim S H, Lubec G (2001). Decreased alpha-endosulfine, an endogenous regulator of ATP-sensitive potassium channels, in brains from adult Down syndrome patients. J Neural Transm Suppl (61), 1-9. 15. Dou J, Cui C, Dufour F, et al. (2003). Gene expression of a-endosulfine in the rat brain: correlative changes with aging, learning and stress. Journal of Neurochemistry 87, 1086-1100. 16. Xu X L, Ma J Q (1998). Biological Chemistry for Medicine Science. Beijing: People Hygiene Press, 231,252,783. 17. Fitch R H, Denenberg V H (1998). A role for ovarian hormones in sexual differentiation of the brain. Behav Brain Sci 21(3), 311-327. (Received November 20, 2006
Accepted July 17, 2007)
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Effects of Nonylphenol on Brain Gene Expression Profiles in F1 Generation Rats1 YIN-YIN XIA, PING ZHAN2, AND YANG WANG Department of Environment Hygiene, Chongqing University of Medical Sciences, Chongqing 400016, China
Objective To explore the effects of nonylphenol on brain gene expression profiles in F1 generation rats by microarray technique. Methods mRNA was extracted from the brain of 2-day old F1 generation male rats whose F0 female generation was either exposed to nonylphenol or free from nonylphenol exposure, and then it was reversely transcribed to cDNA labeled with cy5 and cy3 fluorescence. Subsequently, cDNA probes were hybridized to two BiostarR-40S cDNA gene chips and fluorescent signals of cy5 and cy3 were scanned and analyzed. Results Two genes were differentially down-regulated. Conclusion Nonylphenol may disturb the neuroendocrine function of male rats when administered perinatally. Key words: Nonylphenol; cDNA microarray; Brain tissue
the effects of NP on the early brain development of F1 male rats by cDNA microarray, and to testify the hypothesis that NP has the potential to adversely affect hormonal status.
INTRODUCTION Nonylphenol (NP) is generated from alkylphenol ethoxylates that are widely used in the production of plastics, textiles as detergents, paints, pesticides, and cosmetics[1]. NP has adverse effects on the development of reproductive tract of the male animals which were exposed to it perinatally[2], including reduced size of testes, decreased sperm production, cryptorchidism, and reduced reproductive organ weights. On the other hand, no effects of NP on male reproduction have been found[3]. It was also reported that NP possesses the estrogenic property in vitro and in vivo systems[4]. Brain is the source of behavior, thoughts, feelings, and experiences. If a rodent with functional male genitals is deprived of androgens immediately after birth, its male sexual behavior will be reduced, and more female sexual behaviors will take place instead. These lifelong effects of early exposure to sex hormones are characterized by ‘organizational’ changes because they appear to alter brain function permanently during a critical period in prenatal or early postnatal development. Administering the same sex hormones at later stages or in adults has no such similar effects[5]. The aim of the present study was to investigate
MATERIALS AND METHODS Animals Ten adult male Sprague-Dawley rats (300-350 g) and twenty adult female Sprague-Dawley rats (230-260 g) were purchased from Laboratory Animal Center (West China Center of Medical Sciences), Sichuan University. The animals were housed in a temperature-controlled room with free access to under running water in a 12 h/ dark and light cycle. The F0 rats were housed for 1 week prior to mating. Female rats and male F0 rats were mated with 2:1 proportion overnight and the day when copulatory plugs were found was designated as GD-0. The GD-0 rats were randomly assigned to either treatment or control group. Rats in treatment group were daily given 200 mg/kg NP by oral gavage from GD 7 to PND 2 (day of birth=postnatal day 0), while those in control group received an equivalent volume of corn oil. The filial rats procreated from pregnant F0 rats were denominated first generation rats (F1).
1
This research was supported by the Innovation Fund of Chongqing University of Medical Sciences (No. 200417). Correspondence should be addressed to Ping ZHAN. Tel: 86-23-68485803. Fax: 86-23-68485008. E-mail: [email protected] Biographical note of the first author: Yin-Yin XIA, M. D, female, born in 1978, majoring in endocrine disrupting chemicals. 2
0895-3988/2008 CN 11-2816/Q Copyright © 2008 by China CDC 1
2
XIA, ZHAN, AND WANG
Tissue Samples Brain tissue samples were obtained from PND-2 F1 rats, quickly frozen in liquid nitrogen for RNA extraction. Chip Preparation Four thousand and ninety-six target cDNA clones were used in the rat cDNA chip (provided by Biostar Genechip Inc., Shanghai, China). These genes were amplified by PCR using universal primers and purified with standard method. The quality of PCR was monitored by agarose electrophoresis. Target genes were dissolved in 3×SSC spotting solution, and spotted on silylated slides (Telechem, Inc., USA) by Cartesian 7500 spotting robotics (Cartesian, Inc., USA). Each target gene was dotted twice. After spotting, the slides were hydrated (2 h), dried (0.5 h, room temperature), cross-linked under UV light and treated with 0.2% SDS, H2O and 0.2% NaNBH4 for 10 minutes, respectively. The slides were then dried again for use. Probe Preparation Total RNA in NP-treated and control F1 rat brain tissues was extracted as previously described[6]. The tissues were removed from liquid nitrogen and ground into tiny powder in a ceramic mortar, to which liquid nitrogen was added, and homogenized in D-solution plus 1% mercaptoethanol. After centrifugation, the supernatant was extracted twice with phenol: chloroform (1:1), then once with NaAC and acidic phenol: chloroform (5:1). The aqueous phase was precipitated by equal volumes of isopropanol. The precipitate was centrifuged and dissolved with Millie-Q H2O. After purification by a LiCl precipitating method, UV analysis and electrophoresis detection showed the good quality of purified RNA. mRNA was isolated and purified using an Oligotex mRNA Midi kit (QIAGEN, Inc. USA). The fluorescein-labeled cDNA probe was prepared through retro-transcription and purified, using the method of Schena. cDNA probes from brain tissue of control F1 rats were specifically labelled with Cy3-dUTP and those from brain tissue of NP-treated F1 rats were labelled with with Cy5-dUTP. The probes were mixed (Cy3-dUTP control + Cy5-dUTP ZP-1, Cy3-dUTP control + Cy5-dUTP ZP-2), precipitated by ethanol, and resolved in 20 μL hybridization solution (5×SSC+0.2% SDS). Hybridization and Washing Hybridizing probe and two same chips were denatured in a 95℃ bath for 5 minutes. The probe
was added onto the chips and covered with glass. The chips were hybridized in a sealed chamber at 60℃ for 15-17 h. After the cover glasses were removed, the slides were washed in a solution of 2×SSC+0.2% SDS, 0.1×SSC+0.2% SDS and 0.1% SSC, 10 minutes each, respectively, then dried at room temperature. Fluorescence Scanning and Analysis The chips were scanned with a Scan Array 3000 scanner (General Scanning Inc., USA). The overall intensity of Cy3 and Cy5 was normalized and corrected by a coefficient according to the location ratios of the 40 housekeeping genes. The acquired image was analyzed by ImeGene 3.0 software. Intensity of the fluorescent signal and each ratio of Cy3 to Cy5 were compared. The data were obtained on an average of two repeated spots. The criteria for screening the differentially expressed genes were defined as follows. The absolute value of the natural logarithm of the signal ration of Cy5/Cy3 was greater than 0.69 (gene expression change >2 folds), one of the signal values of Cy3 and Cy5 was greater than 600, and the results of PCR were good. RESULTS The fluorescent scanning profile of gene expression is shown in Fig. 1. Cy3-labeled cDNA probes from normal F1 rat brain tissues and Cy5-labeled cDNA probes from NP-treated F1 rat brain tissues, ZP-1 and ZP-2, were hybridized through microarray. The red spot indicated the upregulated genes, the green spot indicated the down-regulated genes, and the yellow spots indicated genes which had similar expression between the treated F1 rat brain tissues and normal tissues. The scatter plots, plotted from Cy3 and Cy5 fluorescent signal values, revealed a dispersed distribution pattern. Most spots were found along an almost 45° diagonal line. Red spots showed signal differences between 0.5 to 2.0 folds. Certain yellow spots were distributed away from the 45° diagonal line, indicating the existence of many differentially expressed genes. The difference in these signals between control and NP-treated groups was greater than 2.0 folds or less than 0.5 folds (beyond the range of 0.5-2.0 folds) (Fig. 2). Since red indicates the signal difference between 0.5 to 2.0 folds, while yellow represents the signal difference beyond the range of 0.5-2.0 folds, these yellow spots located away from the diagonal line indicated the existence of several differentially expressed genes caused by NP treatment, as shown in Fig. 2.
EFFECTS OF NONYLPHENOL ON BRAIN GENE EXPRESSION
3
FIG. 1. Overlay of hybridizing signals labelled by two-color fluorescence on the gene chip ZP-1 (left), and ZP-2 (right).
FIG. 2. Scatter plots of gene expression pattern, ZP-1(left), and ZP-2 (right).
Seven genes were differentially expressed on ZP-1 gene chip, indicating the altered gene expression caused by NP treatment. The expression of all these genes was down-regulated by 2 folds (ratio<0.5) (Table 1). Among these 7 genes, 2 encoded proteins involved in metabolism of substance and energy, namely stearyl-CoA desaturase (SCD) and glutathione
s-transferase M5. Another 4 genes encoded proteins involved in cell structures and communications, namely endosulfine alpha (Ensa), protein tyrosine phosphatase, receptor type, W (Esp), protein tyrosine phosphatase 2E (PTP2E), actin-related protein 3 (Arp3). Cyp8b1 gene was shown to be a member of cytochrome P450 superfamily.
TABLE 1 List of Differential Expressed Genes, ZP-1 Genbank_ID
Gene Name
Ratio
J02585
Stearyl-CoA Desaturase (SCD)
0.321
U86635
Glutathione s-transferase M5
0.415
Endosulfine alpha (Ensa)
0.420
NM_033099
Protein Tyrosine Phosphatase, Receptor Type, W (Esp)
0.451
NM_031241
Cytochrome P450, 8b1, Sterol 12 alpha-hydrolase (Cyp8b1)
0.488
Protein Tyrosine Phosphatase 2E (PTP2E)
0.490
Actin-related Protein 3 (Arp3)
0.492
NM_021842
U17971 AF307852
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XIA, ZHAN, AND WANG
Sixteen differentially expressed genes were detected on ZP-2 gene chip. Among them, 3 genes were up-regulated by>2.0 folds (ratio>2), while 13 genes were down-regulated by >2.0 folds (ratio<0.5), as indicated in Table 2. Among the 16 genes, 5 encoded proteins involved in metabolism of substance and energy, namely stearyl-CoA desaturase (SCD), liver glycogen phosphorylase (Pygl), peroxisomal 2,4-dienoyl CoA reductase (px-2,4-DCR), succinate dehydrogenase complex, subunit A (SDHA), gonadotropin-regulated long chain acyl-CoA synthetase
(GR-LACS). Another 6 genes encoded proteins involved in cell structures and communications, namely zonula occludens 2 protein (ZO-2), endosulfine alpha (Ensa), ERM-binding phosphoprotein (LOC59114), glutamate receptor (AMPA2), integral membrane-associated protein 1 (Itmap1), transglutaminase 1 (Tgm1). Three genes were found to be members of cytochrome P450 superfamily, namely CYP3A3, CYP4B1, and CYP2C23. The rest were found to be some unsorted genes namely E5E antigen, sarcomeric muscle protein.
TABLE 2 List of Differentially Expressed Genes, ZP-2 Genbank_ID
Gene Name
J02585 NM_013105
Ratio
Stearyl-CoA Desaturase (SCD)
0.236
Cytochrome P450, Subfamily IIIA, Polypeptide 3 (Cyp3a3)
0.247
U75916 NM_022268 NM_021842 AF021854
Zonula Occludens 2 Protein (ZO-2)
0.317
Liver glycogen Phosphorylase (Pygl)
0.330
Endosulfine alpha (Ensa)
0.403
Peroxisomal 2,4-dienoyl CoA Reductase px-2,4-DCR#1
0.443
D37934
5E5 Antigen
0.450
NM_021594
ERM-binding Phosphoprotein (LOC59114)
0.452
NM_017261
Glutamate Receptor, Ionotropic, AMPA2 (alpha 2) (Gria2)
0.454
NM_054005
Integral Membrane-associated Protein 1 (Itmap1)
0.461
AB072907
SDHA mRNA for Flavoprotein Subunit of Succinate-ubiquinone Reductase
0.479
AF208125
Gonadotropin-regulated Long Chain Acyl-CoA Synthetase (GR-LACS)
0.484
NM_031659
Transglutaminase 1, K Polypeptide (Tgm1)
0.485
NM_016999
Cytochrome P450, Subfamily IVB, Polypeptide 1 (Cyp4b1)
2.029
NM_031839
Arachidonic Acid Epoxygenase (Cyp2c23)
2.258
NM_057191
Sarcomeric Muscle Protein (Sarcosin)
2.700
When exposed to NP, statistically significant differential expression was found in 2 genes: ZP-1 and ZP-2. These 2 genes were both suppressed (ratio <0.5), the immediate toxic response appeared to be mediated by the transcriptional regulation of the common genes (Table 3). Two genes, SCD and Ensa, were found to be
differentially expressed simultaneously on both ZP-1 and ZP-2 chips, as shown in Table 3. Both of them were down-regulated (ratio<0.5), indicating that immediate toxic response after nonylphenol exposure could be mediated by the transcriptional regulation of common genes.
TABLE 3 List of Differentially Expressed Genes, ZP-1 and ZP-2 Genbank_ID J02585
Gene Name
Ratio (ZP-1)
Ratio (ZP-2)
Stearyl-CoA Desaturase (SCD)
0.321
0.236
Endosulfine alpha (Ensa)
0.420
0.403
NM_021842
DISCUSSION The study was to characterize the immediate biological responses of F1 male rat brain tissue to
toxic dose of NP and to use this information to develop biomarkers for its toxicity[7]. We used DNA microarray technology to detect NP-induced alterations in gene expression during early brain
EFFECTS OF NONYLPHENOL ON BRAIN GENE EXPRESSION
development of F1 generation male rats. These 23 genes were differentially expressed, involving 7 genes of ZP-1 gene chip and 16 genes of ZP-2 gene chip, representing a broad range of mRNAs encoding diversified functionalities, including metabolism of substance and energy, cell structures, and communications, such as receptors and signaling molecules that regulate the electrophysiological properties of neurons, action maintaining the structural integrity of neurons, as factors regulating fat and protein degradation. Among these 23 genes, however, only 2 were differentially expressed simultaneously on both ZP-1 and ZP-2 chips: Stearyl-CoA desaturase (SCD) gene, endosulfine alpha (Ensa). SCD is consisted of 358 amino acids, of which 62% are hydrophobic amino acids, corresponding to a molecular mass of 41 400 daltons[8]. SCD is a rate limiting enzyme in the biosynthesis of monounsaturated fats and is expressed in the liver, kidneys, brain, and adipose tissue. Oleic acid, a principal product of SCD, is the major unsaturated fatty acid in lipid stores of human adipose tissue and phospholipids in red blood cell membrane. The ratio of stearic acid to oleic acid has been implicated in the regulation of cell growth and differentiation through effects on cell-membrane fluidity and signal transduction[9]. In this study, the down-regulated SCD gene expression in brain tissue of NF-exposed PND-2 F1 rats suggested that NP could affect neuron growth and differentiation by interfering with the biosynthesis of unsaturated fatty acids. Leptin is recognized as the central mediator in an endocrine circuit-regulated energy homeostasis. Cohen et al.[10] found that SCD is specifically repressed during leptin-mediated weight loss, and that adiposity is significantly reduced in mice lacking SCD. These findings indicate that SCD plays an important role in some aspects of metabolic syndrome, suggesting that NP may be associated with energy homeostasis indirectly and a risk factor for metabolic syndrome. Ensa, first isolated from ovine brain as a possible endogenous ligand for sulfonylurea receptors, is expressed in brain, muscle, and endocrine tissues. It is a 121-amino acid-protein with a total molecular mass of 19 kDa, as well as a member of a highly conserved cAMP-regulated phosphoprotein (ARPP) family[11]. The sulphonylurea receptor is physically associated with a pore-forming subunit (Kir 6.2) to constitute the ATP-dependent potassium channel (KATP)[12]. By binding to SUR, Ensa induces closure of KATP channels on the pancreatic β-cells, leading to membrane depolarization, which, in turn, activates
5
voltage-gated L-type Ca2+-channels. The resulting calcium influx triggers insulin release[13]. Ensa is able to block KATP channels not only on the pancreatic β-cells, but also on brain, while the neurobiological role of alpha-endosulfine has not been studied yet. Kim and Lubec[14] examined the expression levels of Ensa in frontal cortex and cerebellum from patients with Down syndrome (DS), and found that considerably decreased Ensa could result in the continuous opening of KATP channels and the subsequent decrease of neurotransmitter release associated with cognition. Dou et al.[15] described that the majority of Ensa genes are expressed in pyramidal neurons of rat brain, which represents the principal excitatory neurons in various brain regions, and swimming-related stress causes persistent up-regulation of Ensa gene expression in several brain regions. In the present study, the down-regulated expression of Ensa gene in brain tissue of NP-exposed PND-2 F1 rats suggests that NP affects the level of insulin by decreasing the expression of Ensa. The decreased Ensa expression results in the continuous opening of KATP channels in brain, inhibits the depolarization of the cell membrane and the calcium influx, and regulates the release of neurotransmitters, such as acetylcholine. Consequently, it is likely that NP disturbs the release of pituitary hormones, such as gonadotropin releasing hormone. This is one of the probable mechanisms that NP impairs neuroendocrine functions by disrupting the synthesis and metabolism of steroid hormone. The response of organs to exposure of NP is often a combined response of various cell types, tissues, and organs within an individual. As brain plays an important role in regulating the metabolism and systemic function, proteins in brain tissue encoded by a specific gene can regulate functions of multiple systems, and proteins encoded by multiple genes regulate the functions of a specific system[16]. Fitch et al. reported[17] that mammalian sexual differentiation is primarily mediated by testicular androgens, and that exposure to androgens in early life leads to a male brain as defined by neuroanatomy and behavior. For F1 male rats, the sensitive period of sexual differentiation extends at least through the perinatal period (roughly from embryonic day 17 through postnatal days 8-10 in rodents). It is likely that exposure to NP during this period may disrupt homeostasis of steroid hormones, influence brain development and sexual differentiation of male rats. In conclusion, nonylphenol may disturb the neuroendocrine function of male rats when administered perinatally. The gene expression
6
XIA, ZHAN, AND WANG
profiles derived from the present study provide insights into potential biomarkers of perinatal NP exposure and possible mechanisms of NP in disrupting neuroendocrine functions. ACKNOWLEDGEMENTS We thank Dr. Hao ZHANG, Department of Environment Hygiene, West China Center of Medical Sciences, Sichuan University, for providing nonylphenol and invaluable information, and Dr. Yuan-Feng LI, Tongji Medical College of Huazhong University of Science and Technology, for the strong support in data collection and manuscript preparation. REFERENCES 1. Naylor C G, Mierure J P, Weeks J, et al. (1992). Alkylphehol ethoxylates in the environment. J Am Oil Chemists 69, 695-703. 2. Boockfor F R, Blake C A (1997). Chronic administration of 4-tertoctylphenol to adult male rats causes shrinkage of the testes and male accessory sex organs, disrupts spermatogenesis, and increases the incidence of sperm deformities. Biol Reprod 57, 267-277. 3. Chapin R E, Delaney J, Wang Y, et al. (1999). The effects of 4-nonylphenol in rats: A multigenerational reproduction study. Toxicol Sci 52, 80-91. 4. Jobling M A, Samara V, Pandya A, et al. (1996). Recurrent duplication and deletion polymorphisms on the long arm of the Y chromosome in normal males. Hum Mol Genet 5, 1767-1775. 5. Kimura D (1992). Sex differences in the brain. Sci Am 267(3), 118-125.
6. Peng X L, Yuan H Y, Xie Y, et al. (1998). Experimental technique of gene engineering. HuNan Science and Technique Issue Agency, Changsha, pp. 197-199. 7. Kaiser S, Nisenbaum L K (2003). Evaluation of common gene expression patterns in the rat nervous system. Physiol Genomics 16(1), 1-7. 8. Thiede M A, Ozols J, Strittmatter P (1986). Construction and sequence of cDNA for rat liver stearyl coenzyme A desaturase. J Biol Chem 261(28), 13230-13235. 9. Lin Z, Lan G E, Satish P, et al. (1999). Human stearoyl-CoA desaturase : alternative transcripts generated from a single gene by usage of tandem polyadenylation sites. Biochem J 340, 255-264. 10. Cohen P, Ntambi J M, Friedman J M (2003). Stearoyl-CoA desaturase-1 and the metabolic syndrome. Curr Drug Targets Immune Endocr Metabol Disord 3(4), 271-280. 11. Virsolvy A, Smith P, Bertrand G, et al. (2002). Block of Ca(2+)-channels by alpha-endosulphine inhibits insulin release. Br J Pharmacol 135(7), 1810-1818. 12. Inagaki N, Gonoi T, Clement JP 4th, et al. (1995). Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270(5239), 1166-1170. 13. Ashcroft S J, Ashcroft F M (1990). Properties and functions of ATP-sensitive K-channels. Cell Signal 2(3), 197-214 14. Kim S H, Lubec G (2001). Decreased alpha-endosulfine, an endogenous regulator of ATP-sensitive potassium channels, in brains from adult Down syndrome patients. J Neural Transm Suppl (61), 1-9. 15. Dou J, Cui C, Dufour F, et al. (2003). Gene expression of a-endosulfine in the rat brain: correlative changes with aging, learning and stress. Journal of Neurochemistry 87, 1086-1100. 16. Xu X L, Ma J Q (1998). Biological Chemistry for Medicine Science. Beijing: People Hygiene Press, 231,252,783. 17. Fitch R H, Denenberg V H (1998). A role for ovarian hormones in sexual differentiation of the brain. Behav Brain Sci 21(3), 311-327. (Received November 20, 2006
Accepted July 17, 2007)