The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.4, 605-608, 2010
605
Toxicogenomics/proteomics Report
Estrogenic effects of o,p’-DDT exposure in Japanese medaka (Oryzias latipes) Masaya Uchida1,2, Hiroshi Nakamura2, Yoshihiro Kagami2, Teruhiko Kusano2 and Koji Arizono3 Graduate School of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan 2Ecogenomics, Inc., 1-1 Hyakunenkouen, Kurume, Fukuoka 839-0864, Japan 3Faculty of Environmental and Symbiotic Sciences, Prefectural University of Kumamoto, 3-1-100 Tsukide, Kumamoto 862-8502, Japan 1
(Received March 1, 2010; Accepted May 6, 2010)
ABSTRACT — The persistent pesticide 1,1,1-trichloro-2-(2-chlorophenyl)-2-(4-chlorophenyl)ethane (o,p’-DDT) has been implicated as an endocrine-disrupting chemical. In this study, we performed DNA microarray analysis to assess hepatic gene expression in male Japanese medaka (Oryzias latipes) exposed to 1 ppb and 100 ppb o,p’-DDT for 48 hr. Results showed that 1 ppb o,p’-DDT induced the expression of choriogenins (chgH, chgL, and chgH minor), and 100 ppb induced the expression of vitellogenins (vtgI and vtgII) and estrogen receptor alpha. These genes showed considerably high up-regulation among the genes assayed and showed good dose-dependency. Thus, in this study the female hormone-like endocrinedisrupting effect of o,p’-DDT at gene expression level was clearly observed in Japanese medaka. Key words: o,p’-DDT, Japanese medaka (Oryzias latipes), DNA microarray
INTRODUCTION Dichloro-diphenyl-trichloroethane (DDT) is one of the most well-known synthetic organochlorine pesticides used for the prevention of malaria (ICPS,1979), which compound structure permits several different isomeric forms, and has been implicated as an endocrine disrupting chemical (Arcand-Hoy and Benson, 1997). By the Stockholm Convention, which was ratified in May 2004 by 50 nations, initiatives were taken to minimize production, import, export, use, and release of twelve persistent organic pollutants (POPs) (UNEP-Chemicals, 2004). Of these Stockholm Convention POPs, nine (aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, hexachlorobenzene, mirex, and toxaphene) were in fact organochlorine pesticides, and DDT was included. The moratorium on DDT use unfortunately resulted in an increase in the number of human deaths from malaria, and in response South Africa appealed for a “limited and controlled” application of DDT to eradicate the malaria-causing pyrethroid-resistant mosquito, Anopheles funestus (Bouwman, 2000). In 2006, the World
Health Organization (WHO) and the US Agency for International Development (USAID) decided to endorse indoor DDT spraying to control malaria (WHO, 2006). DDT is highly toxic to fish; the 96 hr LC50 (static test) ranges from 1.5 to 56 ppb (largemouth bass and guppy, respectively), and juvenile fish seems to be more susceptible than adult fish in the same species (IPCS, 1989). In spite of these facts, there is few toxicological research focusing on comprehensive molecular responses (differential gene expression) to DDT exposure (Shutoh et al., 2009). In present study, male-to-female sex reversal occurred in 86% of genetic males after exposure to 227 ± 22 ng/egg o,p’-DDT employing microinjection (Edmunds et al., 2000). Effects of o,p’-DDT on gene expression has rarely been discussed so far. Thus, the aim of this study was to investigate toxicologically significant effects of o,p’-DDT exposure on gene expression in Japanese medaka (Oryzias latipes) using DNA microarray.
Correspondence: Masaya Uchida (E-mail:
[email protected])
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MATERIALS AND METHODS Chemicals The organochlorine o,p’-DDT (Wako pure Chemical Industries, Osaka, Japan : Cas No. 789-02-6) was dissolved in Methanol. The methanol of at least 99.8% purity was purchased from Wako pure Chemical Industries. Fish Adult Japanese medaka (3-month-old males of the orange-red strain, i.e., weight 266.1 ± 36.8 mg, length 29.80 ± 1.2 mm) were obtained from National Institute for Environmental Studies (Ibaraki, Japan). They selected from a brood stock at Ecogenomics, Inc. (Fukuoka, Japan) for more than 7 years, and they were maintained in a 5-l glass aquarium with flow-through system at 24 ± 2°C and a 16 hr : 8 hr light:dark cycle. Fish were fed twice daily with brine shrimp (within 24 hr post-hatch) (El-Alfy and Schlenk, 2002). Exposure design Adult male medaka were exposed to 1 ppb and 100 ppb o,p’-DDT in 2-l glass aquarium for 48 hr without being fed. In each experiment, control fish were exposed to vehicle (100 ppb methanol). After the exposure, liver was surgically removed and immediately immersed in RNAlater solution (Sigma-Aldrich, St. Louis, MO, USA) in order to inhibit RNA degradation. Medaka DNA microarray gene expression analysis Total RNA was extracted from the liver with an RNeasy Mini Kit (Qiagen, Hilden, Germany). Integrity of the total RNA was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and the qualified total RNA samples were used in the further DNA microarray analyses. For DNA microarray hybridization, antisense RNA (aRNA) was our choice, prepared by Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion, Austin, TX, USA), and a fluorescent dye, Cy5 (GE Healthcare, Little Chalfont, UK), was used to label the aRNA samples.. DNA microarray hybridization and analyses were carried out with EG Medaka-750 microarray (Ecogenomics, Inc.), that contained 835 medaka cDNA probes (300 ± 25 bases in length) and each of them were spotted in triplicate. The Cy5-labeled aRNA samples were hybridized to the microarray probes in 40 μl of hybridization solution in a moist chamber for 16 hr at 42°C. The hybridization solution contained 50% formamide (Wako Pure Chemical Industries), 5 × saline-sodium citrate (SSC; Sigma-Aldrich), and 0.5% sodium dodecyl sulfate (SDS; Vol. 35 No. 4
Ambion). After the hybridization, the microarrays were washed by 1 × SSC/0.2% SDS, 0.1 × SSC/0.2% SDS, and 0.1 × SSC wash solutions in such an order. Then, the microarrays were scanned with FLA-8000 Fluorescent Image Analyzer (Fujifilm, Tokyo, Japan) at 10 μm-resolution, and expression signals of the 835 genes were quantified. Statistical analysis Expression signal data collected from each of the 835 microarray gene probes were normalized to that of ribosomal protein L7 mRNA. Subsequently, the normalized data were analyzed statistically by t-test with offset correction and p < 0.05 significance cutoff to obtain significant change in each gene’s expression ratio between exposure and control. Quantitative real-time polymerase chain reaction analysis Results of the microarray analysis were validated by quantitative real-time polymerase chain reaction (qRT-PCR) analysis. Template cDNA was synthesized from the same set of total RNA samples that were used for the DNA microarray analysis with Moloney murine leukemia virus (MMLV) reverse transcriptase (ReverTra Ace; TOYOBO, Osaka, Japan) with oligo-dT primer (Ambion). The qRT-PCR was performed with a SYBR Green Realtime PCR Master Mix Plus Kit (TOYOBO) using ABI7300 Realtime PCR System (Applied Biosystems, Foster City, CA, USA). Relative expression levels were obtained, and the expression level of each gene was normalized to that of ribosomal protein L7 mRNA (Zhang and Hu, 2007). All experiments were performed in triplicate for each sample. RESULTS AND DISCUSSION We used DNA microarray analysis to assess molecular toxicological effects of o,p’-DDT exposure in the liver of adult Japanese medaka. Male medaka were exposed to two concentrations of o,p’-DDT (1 and 100 ppb) for 48 hr, and mRNA expression profiles in their livers were analyzed. We identified statistically significant (p < 0.05) up-regulation (expression ratio of exposed to control > 3) in 8 genes responding to 1 ppb o,p’-DDT and 25 genes responding to 100 ppb (Table 1). The greatest up-regulation in response to 100 ppb o,p’-DDT was observed in choriogenins (chgH, chgL, and chgH minor), vitellogenins (vtgI and vtgII), and estrogen receptor alpha (Table 2). In medaka, choriogenins are egg envelope precursor proteins that are synthesized in the liver of spawning
607 Gene expression in Japanese medaka exposed to DDT
Table 1. List of genes up-regulated in response to o,p’-DDT exposure in Japanese medaka Accession# 1 ppb exposure AB070901 AB030069 AB030072 AB026950 AU177207 BJ008588 BJ530112 AJ457486
Gene name
Ratio
estrogen receptor beta cyclin B1 cyclin B2 HOX A2B low density lipoprotein receptor related protein - human Pyridoxal kinase putative (Anoplopoma fimbria) Pleuronectes platessa partial mRNA for transforming growth factor all_halibut.250.C2 mRNA (Hippoglossus hippoglossus)
100 ppb exposure D89609 choriogenin H AF500194 choriogenin L AB025967 choriogenin H minor AB070901 estrogen receptor beta AU180885 apoptosis inhibitor hiap-2 (human) AV670049 metastasis-associated protein mta-1 (rat) AB001572 hepatocyte nuclear factor-3beta gene (Me-HNF3B) BJ490983 jun D proto-oncogene (bovine) BJ492759 Cell division control protein 42 (Anoplopoma fimbria) BJ009539 serine hydroxymethyltransferase 1 (Danio rerio) AB033491 estrogen receptor alpha AB064320 vitellogenin I AB074891 vitellogenin II AU180054 vitellogenin A2 precursor (African clawed frog) BM309828 vitellogenin III precursor (Danio rerio) AF359589 insulin-like growth factor binding protein 2 AB001603 KFH-G AB026950 HOX A2B BJ012673 HOX Ca AB030072 cyclin B2 AU240622 protein kinase cdc2-related PITALRE (human) BJ001720 glutamyl-prolyl-tRNA synthetase BJ012241 Heterogeneous nuclear ribonucleoprotein A/B putative (Salmo salar clone) BJ526571 mitogen-activated protein kinase-activated protein kinase 2a (Danio rerio) AJ457486 all_halibut.250.C2 mRNA (Hippoglossus hippoglossus) Up-regulation was defined as an expression ratio of exposed to control > 3 and < 0.5. a) SD: standard deviation b) Raw data is presented for samples for which the control signal was zero.
females in response to estrogen, and they have been used as biomarkers for monitoring estrogenic chemicals in the aquatic environment (Sugiyama et al., 1999). Also, vitellogenins are frequently used as biomarkers for assessing the effects of estrogenic chemicals (Iguchi et al., 2006). Up-regulation of these estrogen-responsive genes by o,p’DDT showed dose-dependent induction similar to the one induced by estrogen and estrogen-like chemicals (Table
±S.D.a)
8.13 21.07 >3 b) >3 b) >3 b) >3 b) >3 b) >3 b)
0.67 5.84 -
29.74 6.49 64.58 3.41 3.91 5.30 9.32 8.62 7.70 3.34 >3 b) >3 b) >3 b) >3 b) >3 b) >3 b) >3 b) >3b) >3 b) >3 b) >3 b) >3 b) >3 b) >3 b) >3 b)
4.61 0.34 3.09 0.51 0.47 0.65 1.07 0.89 0.35 0.32 -
2). These sets of DNA microarray data were consistent with those obtained from qRT-PCR validation analysis. o,p’-DDT at concentration of 1 ppb also induced the expression (expression ratio exposed to control = 7.2) of a gene similar to human dexamethasone-induced ras-related protein 1, which is expressed in a variety of tissues including heart, cardiovascular tissues, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, gastroinVol. 35 No. 4
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Table 2. Comparision of expression of estrogenic genes as determined by DNA microarray and qRT-PCR Ratio 1 ppb 100 ppb microarray qRT-PCR microarray qRT-PCR D89609 choriogenin H 6.8 8.6 30 308 AF500194 choriogenin L 1.6 0.4 6.5 116 1.4 >10 b) 65 AB033491 estrogen receptor alpha n.d. a) 9.1 >100 b) 32844 AB064320 vitellogenin I n.d. a) AB074891 vitellogenin II n.d. a) 19 >100 b) 152664 b) BJ490945 dexamethasone-induced Ras-related protein 1 >3 7.2 n.d. a) 2.1 AB025967 choriogenin H minor 2.2 2.3 65 310 41 >100 b) 53726 AU180054 vitellogenin A2 precursor (African clawed frog) n.d. a) 56 >100 b) 108450 BM309828 vitellogenin III precursor (Danio rerio) n.d. a) Values indicate ratio of gene expression in DDT-exposed medaka to that in control medaka. qRT-PCR, quantitative real-time polymerase chain reaction. a) n.d.: not detected b) Raw data is presented for samples for which the control signal was zero. The magnification is calculated from lower detection limit. Accession#
Gene Name
testinal, and reproductive tissues (Su et al., 2004). The function of this gene in medaka is unknown, but it may provide information as a novel biomarker due to its sensitivity to the low concentration of o,p’-DDT. The possibility of using dexamethasone-induced ras-related protein 1 as a biomarker will be investigated in future experiments. In conclusion, the results of this study indicated that o,p’-DDT had distinctive effects on the endocrine system and the liver mRNA expression of male Japanese medaka. ACKNOWLEDGMENT This work was supported by a Pesticide Environment Study Subsidy from the Pesticide Science Society of Japan. REFERENCES Arcand-Hoy, L.D. and Benson, W.H. (1997): Fish reproduction: an ecologically relevant indicator of endocrine disruption. Environ. Toxicol. Chem., 17, 49-57. Bouwman, H. (2000): Malaria control and the paradox of DDT. Afr. Environ. Wildlife, 8, 54-56. Edmunds, J.S., McCarthy, R.A., and Ramsdell, J.S. (2000): Permanent and functional male-to-female sex reversal in d-rR strain medaka (Oryzias latipes) following egg microinjection of o,p’DDT. Environ. Health Perspect., 108, 219-224. El-Alfy, A.T. and Schlenk, D. (2002): Effect of 17beta-estradiol and testosterone on the expression of flavin-containing monooxygenase and the toxicity of aldicarb to Japanese medaka, Oryzias latipes. Toxicol. Sci., 68, 381-388. Iguchi, T., Irie, F., Urushitani, H., Tooi, O., Kawashima, Y., Roberts, M., Norrgren, L. and Hutchinson, T.H. (2006): Availability of in vitro vitellogenin assay for screening of estrogenic and antiVol. 35 No. 4
estrogenic activities of environmental chemicals. Environ. Sci., 13, 161-183. IPCS (1979): DDT and its derivatives. Environmental health Criteria 9. IPCS (1989): DDT and its derivatives - Environmental Aspects. Environmental health Criteria 83. Su, A.I., Wiltshire, T., Batalov, S., Lapp, H., Ching, K.A., Block, D., Zhang, J., Soden, R., Hayakawa, M., Kreiman, G., Cooke, M.P., Walker, J.R. and Hogenesch, J.B. (2004): A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl. Acad. Sci. USA, 101, 6062-6067. Sugiyama, H., Murata, K., Iuchi, I., Nomura, K. and Yamagami, K. (1999): Formation of mature egg envelope subunit proteins from their precursors (choriogenins) in the fish, Oryzias latipes: Loss of partial C-terminal sequences of the choriogenins. J. Biochem., 125, 469-475. Shutoh, Y., Takeda, M., Ohtsuka, R., Haishima, A., Yamaguchi, S., Fujie, H., Komatsu, Y., Maita, K. and Harada, T. (2009): Low dose effects of dichlorodiphenyltrichloroethane (DDT) on gene transcription and DNA methylation in the hypothalamus of young male rats: implication of hormesis-like effects. J. Toxicol. Sci., 34, 469-482. UNEP-Chemicals (2004): Stockholm Convention on Persistent Organic Pollution, United Nation Environment Programs Available from: http://www.pops.int/. WHO (2006): Indoor Residual Spraying: Use of Indoor Residual Spraying for Scaling Up Global Malaria Control and Elimination: WHO Position Statement. Geneva: Global Malaria Programme, World Health Organization. Zhang, Z. and Hu, J. (2007): Development and validation of endogenous reference genes for expression profiling of medaka (Oryzias latipes) exposed to endocrine disrupting chemicals by quantitative real-time RT-PCR. Toxicol. Sci., 95, 356-368.
