Abstract
Expressions of the estrogen receptors and vitellogenin in Japanese turtle (Mauremys japonica) in response to petroleum hydrocarbon were studied. A total of 15 male turtles were exposed to 1.00 mg L-1 of a sample of an oil spill, and 15 male and 15 female turtles were served as controls without an oil spill. The transcripts' results demonstrated an increase over time with greater expression of vitellogenin I in males exposed to petroleum with significant differences. In the case of vitellogenin II, the expression was greater than control males, but it was similar to the values of control females. Concerning the estrogen receptor and estrogen receptor , males exposed to oil spill presented higher values at 72 hr than the controls. In conclusion, in the present work, the effect of petroleum as an endocrine disruptor in turtles was demonstrated, and it can be used to identify damages induced by the presence of hydrocarbons in aquatic environments.
Key Words: Endocrine disruptor, Japanese turtle, Petroleum hydrocarbon, Quantitative-PCR, Xenobiotic
Introduction
The exposure to endocrine disruptors (EDCs) of the wildlife is global. The EDCs alter hormone balance, some at low doses and by various mechanisms. Hormone levels vary with age, sex, and time, so to estimate the consequences of EDCs exposure, each species' hormonal pattern should be taken.1-3 Four major routes of EDCs action can be distinguished: a) Binding to activation of androgenic and estrogenic receptors; b) Blockage of estrogen receptors without activation; c) Modification of hormone metabolism; d) Modification of the hormones receptors number.4,5 Many environmental chemicals affecting endocrine systems are persistent and bio-accumulative, such as halogenated organic, pesticides, drugs, hydrocarbons, and hydrocarbons derivates.6,7 To understand the links between exposure and adverse effects, the classification of the molecular bases of EDCs and endogenous estrogen needs to be clarified in the development agencies.8 A sensitive method detecting the exposure and the effects of EDCs is by monitoring the expression of the genes involved.9,10
In this study, quantitative-PCR was used to determine the mRNA involved in the gene expression of vitellogenin (VTG) in Japanese turtles (Mauremys japonica) exposed to petroleum hydrocarbon.
Materials and Methods
Experimental design and sampling. Japanese turtles were purchased from Aquapolis Acuario (Campeche, Mexico), maintained in a glass aquarium of 20.00 L with up to 3.00 cm chlorine-free hard water for three weeks11 and fed with food for reptiles (Wardley, Walmart, USA) ad libitum three times a day. All procedures were following the guidelines approved by Biological Ethics Committee of EPOMEX Institute, Campeche University (NOM-019-92 STPS-1993).The pilot hydrocarbon came from an oil spill that occurred in 2007 in the Gulf of Mexico. The chemical analysis indicated the presence of aliphatic hydrocarbons of C14 to C39 (69.96%) and aromatic hydrocarbons (0.524 g g-1) including acenaphthene, acenaphthylene, anthracene, benzo [k] fluoranthene, fluoranthene, fluorene, indeno [1,2,3-cd] pyrene, and phenanthrene.12
Fifteen male turtles were exposed to 1.00 mg L-1 of a sample of the oil spill, and 15 male and 15 female turtles were served as controls without an oil spill. The turtles were placed in hard water. The bioassay was carried out over 72 hr in a static test design according to the criteria of chronic exposure.11 Every 24 hr, five turtles from each treatment were euthanized to extract the liver. The turtles were euthanized by placing in 500 mL bottles having a lid with a tube adapted to supply carbon dioxide for 5 min. Once the unconsciousness was verified, the animals were decapitated.
mRNA expression analysis. Total RNA was isolated from the liver under the manufacturer´s instructions (GeneJET RNA Purification Kit; Thermo Fisher Scientific, Foster City, USA). The RNA was quantified using NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies LLC, Wilmington, USA), and its quality was assessed by the presence of ribosomal bands in ethidium-bromide stained agarose gels. The RNA was diluted to approximately 1.00 mg mL-1 for the reverse transcription-polymerase chain reaction (RT-PCR). The RT-PCR was performed according to the manufacturer’s instructions (TaqMan Reverse Transcription Reagent; Thermo Fisher Scientific, USA). For the relative quantification of gene expression, quantitative-PCR on StepONE Q-PCR equipment (Applied Biosystems, Foster City, USA) was used. The sequences of the primers are shown in Table 1. The PCR conditions were as follows: Initial denaturation at 94.00 ˚C for 30 sec, hybridization at 60.00 ˚C for 30 sec, and extension at 72.00 ˚C for 60 sec. For quantification, -actin gene was used as a reference, and 2CT method was used to calculate the expression.13
Table 1.
Sequences of primer pairs were used in the Q-PCR study.
| Gene Name | Primer Sequences |
|---|---|
| Estrogen receptor ( AB033491 ) | 5´-GTCAGTCGGGTTACTTGGCC-3´ 5´-CATCACCTTGTCCCAACCTG-3´ |
| Estrogen receptor ( AB070901 ) | 5´-GTGGACTCAACTTTCGGC-3´ 5´-CACGTCGCAGCAGGATCTT-3´ |
| Vitellogenin I ( AB064320 ) | 5´-TGGAAAGGCTGARGGGGAAG-3´ 5´-AACTGCAGGCATGGTGAGCC-3´ |
| Vitellogenin II ( AB075891 ) | 5´-GTCTTCAGGAGGTCTTCTTC-3´ 5´-GGTAGACAATGGTATCCGAC-3´ |
| -Actin ( S74868 ) | 5´-AGACCACCTACAGCATC-3´ 5´-TCTCCTTCTGCATTCTGTCT-3´ |
Statistical analysis. Values of parameters were used to detect the interaction between petroleum hydrocarbon and exposure time by two-way ANOVA. If a significant interaction was detected between the main effects, the variable was analyzed using a one-factor ANOVA. If there was a significant difference, F-test was performed using the Statgraphics Centurion X (Statgraphics Technologies, Inc., The Plains, USA).
Results
The transcripts' results showed an increase in the time with greater expression of VTG I in males exposed to petroleum compared to controls with significant differences (p < 0.05; Fig. 1). In VTG II, the increase in exposed males was higher than control males, but it was lower than control females (Fig. 1). For the estrogens receptors and , they presented higher values at 72 hr compared to the controls (p < 0.05; Fig. 2).
Fig. 1.
The relative intensity of vitellogenin I and II (VTG I and VTG II) in the liver of Japanese pond turtle (M. japonica). Different letters indicate significant differences at p < 0.05
Fig. 2.
The relative intensity of estrogen receptors and in the liver of Japanese pond turtle (M. japonica). Different letters indicate significant differences at p < 0.05
Discussion
In oviparous animals, VTG induction is used as a bio-marker to evaluate the presence of estrogenic compounds.14 In the environment, many contaminating compounds are xenoestrogens and have shown estrogenic response in vivo and in vitro.15 In the reptile species, synthesis of estrogen has been observed in other organs aside from ovaries.16 Some authors have reported a small amount of estrogen production in the adrenal gland and liver, resulting in the local synthesis of VTG.17 In this study, effects of endocrine disruption on the VTG synthesis were revealed. In studies carried out in sea turtles (Lepidochelys kempi), VTG has been detected in adult females blood. The VTG is produced in the liver in the presence of estradiol and deposited in the yolks of eggs.18 In juveniles and males, the VTG expression is not common, but in the presence of estrogens or xenoestrogens, the expression is possible. Cheek et al. have observed the VTG production in juveniles of marine turtles exposed to estradiol.18 Keller et al. have studied 400 loggerhead turtles (Caretta caretta), reported an expression of VTG in male and female youth, and concluded that xenoestrogen compounds in the environ-ment might cause this expression.19 In this study, activations of VTG genes and estrogen receptors were observed over time with oil exposure. Smelker et al. have mentioned that the expression of VTG is possible in males in the presence of ECDs in the environment, and it was also verified in the loggerhead turtle (C. caretta).20 Marquez et al. have reported that VTG expression in painted turtle (Chrysemys picta) males is caused by the presence of petroleum and concluded that hydrocarbons affect the aryl hydrocarbon receptors, a gene regulating the genes involved in the development of gonads.21 Rochman et al. have demonstrated that petroleum alters the endocrine system resulting in a permanent effect on the aquatic fauna.22
In conclusion, in this study, the effect of petroleum as an endocrine disruptor in turtles was demonstrated, and it can be used to identify damages induced by the presence of hydrocarbons in the aquatic environment.
Acknowledgments
The authors are thankful to the staff of the Laboratory of Molecular and Microbiology Unit, EPOMEX Institute, Campeche University (Campeche, Mexico).
Conflict of interest
The authors declare no conflict of interest.
References
- 1.Olea N, Fernández MF, Araque P, et al. Prospects for endocrine disruption. [Spanish]. Gac Sani. 2002;16(3):250–256. doi: 10.1016/s0213-9111(02)71670-1. [DOI] [PubMed] [Google Scholar]
- 2.Arukwe A, Goksøyr A. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp Hepatol. 2003;2(1):4. doi: 10.1186/1476-5926-2-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Argemi F, Cianni N, Porta A. Endocrine disruption: environmental perspectives and public health [Spanish] Acta Bioquim Clin L. 2005;39(3):35–41. [Google Scholar]
- 4.Fox JE, Starcevic M, Jones PE, et al. Phytoestrogen signaling and symbiotic gene activation are disrupted by endocrine-disrupting chemicals. Environ Health Perspect. 2004;112(6):672–677. doi: 10.1289/ehp.6456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Tabb MM, Kholodovych V, Grün F, et al. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR) Environ Health Perspect. 2004;112(2):163–169. doi: 10.1289/ehp.6560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Larsson DGJ, Adolfsson-Erici M, Parkkonen J, et al. Ethinyloestradiol – an undesired fish contraceptive? Aquat Toxicol. 1999;45(2-3):91–97. [Google Scholar]
- 7.Hyndman KM, Biales A, Bartell SE, et al. Assessing the effects of exposure timing on biomarker expression using 17β-estradiol. Aquat Toxicol. 2010;96(4):264–272. doi: 10.1016/j.aquatox.2009.11.004. [DOI] [PubMed] [Google Scholar]
- 8.Iguchi T, Watanabe H, Katsu Y. Toxicogenomics and ecotoxicogenomics for studying endocrine disruption and basic biology. Gen Comp Endocrinol. 2007;153(1-3):25–29. doi: 10.1016/j.ygcen.2007.01.013. [DOI] [PubMed] [Google Scholar]
- 9.Yamaguchi A, Ishibashi H, Kohra S, et al. Short-term effects on endocrine-disrupting chemicals on the expression of estrogen-responsive genes in male medaka (Oryzias latipes) Aquat Toxicol. 2005;72(3):239–249. doi: 10.1016/j.aquatox.2004.12.011. [DOI] [PubMed] [Google Scholar]
- 10.Ishibashi H, Yamauchi R, Matsuoka M, et al. Fluorotelomer alcohols induce hepatic vitellogenin through activation of the estrogen receptor in male medaka (Oryzias latipes) Chemosphere. 2008;71(10):1853–1859. doi: 10.1016/j.chemosphere.2008.01.065. [DOI] [PubMed] [Google Scholar]
- 11.Rendón-von Osten J, Ortíz-Arana A, Guilhermino L, et al. In vivo evaluation of three biomarkers in the mosquitofish (Gambusia yucatana) exposed to pesticides. Chemosphere. 2005;58(5):627–636. doi: 10.1016/j.chemosphere.2004.08.065. [DOI] [PubMed] [Google Scholar]
- 12.Rendón- von Osten J. Report on the analysis of petroleum hydrocarbons in water and sediment samples from the Usumacinta platform area, Gulf of Mexico [Spanish] 1st ed. Campeche, Mexico: Autonomous University of Campeche; 2009. pp. ;55–67. [Google Scholar]
- 13.Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
- 14.King-Heiden TC, Spitsbergen J, Heideman W, et al. Persistent adverse effects on health and reproduction caused by exposure of zebrafish to 2,3,7,8-tetra-chlorodibenzo-p-dioxin during early development and gonad differentiation. Toxicol Sci. 2009;109(1):75–87. doi: 10.1093/toxsci/kfp048. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Nakada N, Nyunoya H, Nakamura M, et al. Identification of estrogenic compounds in wastewater effluent. Environ Toxicol Chem. 2004;23(12):2807–2815. doi: 10.1897/03-699.1. [DOI] [PubMed] [Google Scholar]
- 16.Zaccaroni A, Zucchini M, Segatta L, et al. Vitellogenin (VTG) conservation in sea turtles: anti-VTG antibody in Chelonia mydas versus Caretta caretta. Physiol Biochem Zool . 2010;83(1):191–195. doi: 10.1086/648486. [DOI] [PubMed] [Google Scholar]
- 17.Pieau C, Dorizzi M. Oestrogens and temperature-dependent sex determination in reptiles: all is in the gonads. J Endoncrinol. 2004;181(3):367–377. doi: 10.1677/joe.0.1810367. [DOI] [PubMed] [Google Scholar]
- 18.Cheek AO, Brouwer TH, Carroll S, et al. Experimental evaluation of vitellogenin as a predictive biomarker for reproductive disruption. Environ Health Perspect. 2001;109(7):681–690. doi: 10.1289/ehp.01109681. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Keller JM, McClellan-Green PD, Kucklick JR, et al. Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments. Environ Health Perspect. 2006;114(1):70–76. doi: 10.1289/ehp.8143. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Smelker K, Smith L, Arendt M, et al. Plasma vitellogenin in free-ranging loggerhead sea turtles (caretta caretta) of the northwest Atlantic ocean. J Mar Biol. 2014;2014(1):748267. [Google Scholar]
- 21.Marquez EC, Traylor-Knowles N, Novillo-Villajos A, et al. Novel cDNA sequences of aryl hydrocaqrbon receptors and gene expression in turtles (Chrysemys picta and Pseudemys scripta) exposed to different environments. Comp Biochem Physiol C Toxicol Pharmacol. 2011;154(4):305–317. doi: 10.1016/j.cbpc.2011.06.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Rochman , CM , Lewison RL, Eriksen M, et al. Polybrominated diphenyl ethers (PBDEs) in fish tissue may be an indicator of plastic contamination in marine habitats. Sci Total Environ. 2014:476–477: 622-633. doi: 10.1016/j.scitotenv.2014.01.058. [DOI] [PubMed] [Google Scholar]