Article Cite This: Environ. Sci. Technol. XXXX, XXX, XXX−XXX
pubs.acs.org/est
Blood Transcriptomics Analysis of Fish Exposed to Perfluoro Alkyls Substances: Assessment of a Non-Lethal Sampling Technique for Advancing Aquatic Toxicology Research Ignacio A. Rodríguez-Jorquera,*,†,‡,§ R. Cristina Colli-Dula,§,∥ Kevin Kroll,§ B. Sumith Jayasinghe,§ Maria V. Parachu Marco,⊥,#,∇ Cecilia Silva-Sanchez,§ Gurpal S. Toor,○ and Nancy D. Denslow§ †
Centro de Humedales Río Cruces (CEHUM), Universidad Austral de Chile, Independencia 641,Valdivia, Región de los Ríos, Chile Interdisciplinary Ecology Program, School of Natural Resources and Environment, Soil and Water Science Department and § Department of Physiological Sciences & Center for Environmental and Human Toxicology, University of Florida, Gainesville, Florida 32611, United States ∥ Departamento de Recursos el Mar, Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, Unidad Mérida, México ⊥ Laboratorio de Biología Celular y Molecular Aplicada (LBCMA), Instituto de Ciencias Veterinarias del Litoral (ICiVet - Litoral), Universidad Nacional del Litoral (UNL)/Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), CP 3080 Esperanza, Santa Fe, Argentina # Facultad de Humanidades y Ciencias - Universidad Nacional del Litoral, Paraje El Pozo sin nombre, CP 3000 Santa Fe, Argentina ∇ Proyecto Yacaré, Laboratorio de Zoología Aplicada: Anexo Vertebrados (Facultad de Humanidades y Ciencias - Universidad de Nacional del Litoral/MASPyMA), Aristóbulo del Valle 8700, 3000 Santa Fe (Santa Fe) Argentina ○ Department of Environmental Science and Technology, University of Maryland, College Park, Maryland 20742, United States
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S Supporting Information *
ABSTRACT: In contrast to mammals, the blood from other vertebrates such as fish contains nucleated red cells. Using a fathead minnow (Pimephales promelas) oligonucleotide microarray, we compared altered transcripts in the liver and whole blood after exposure to environmentally relevant concentrations of perfluorooctanesulfonic acid (PFOS) and a mixture of seven types of perfluoro alkyl substances (PFAS), including perfluorooctanoic acid (PFOA). We used quantitative polymerase chain reactions and cell-based assays to confirm the main effects and found that blood responded with a greater number of altered genes than the liver. The exposure to PFAS altered similar genes with central roles in a cellular pathway in both tissues, including estrogen receptor α and peroxisome proliferator activator β and γ, indicating that the genes previously associated with PFAS exposure are differentially expressed in blood and liver. The altered transcripts are involved with cholesterol metabolism and mitochondrial function. Our data confirmed that PFAS are weak xenoestrogens and exert effects on DNA integrity. Gene expression profiling from blood samples not related with the immune system, including very-low-density lipoprotein, vitellogenin, estrogen receptor, and thyroid hormone receptor, demonstrated that blood is a useful tissue for assessing endocrine disruption in non-mammalian vertebrates. We conclude that the use of blood for non-lethal sampling in genomics studies is informative and particularly useful for assessing the effects of pollution in endangered species. Further, using blood will reduce animal use and widen the experimental design options for studying the effects of contaminant exposure on wildlife.
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INTRODUCTION Aquatic pollution affects biodiversity at local, regional, and global scales.1 In response to this issue, several techniques have been developed to research the impact of pollution on biota. Recently, genomic tools have improved our understanding of how chemicals impact the environment, revolutionizing both the field of toxicology and the field of ecotoxicology.2 Specifically, transcriptomic analyses have been used to understand the effects of aquatic pollution on biota.3−5 Transcriptomics profiling allows researchers to perform analysis of thousands of genes at a time to identify expression © XXXX American Chemical Society
changes in response to chemical exposures, which can help us better understand the mode of action associated with biological responses to these exposures.2 Despite major advances in techniques in environmental toxicology and ecological risk assessment, improvements in data collection are necessary to understand the effects of Received: Revised: Accepted: Published: A
June 30, 2018 November 15, 2018 December 20, 2018 December 20, 2018 DOI: 10.1021/acs.est.8b03603 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology pollution at the population level. For instance, the duration and consistency of pollutant effects and adaptation and recovery periods collected in laboratory micro- and macromesocosm experiments should be evaluated over time to ensure a realistic response.6 In this context, the development of non-lethal techniques with which to assess pollution effects on the same individual will help advance the understanding and action of pollution. Therefore, the use of blood emerges as an ideal tissue to assess changes in physiology in vertebrates caused by external stressors, particularly the non-mammalian vertebrates that have nucleated red cells and functional mitochondria.7 Advances in genomics approaches, especially for dealing with specific issues affecting human subjects, allow the use of blood as a non-lethal sampling method to analyze gene expression signatures for toxicity determination.8,9 Moreover, blood is a logical choice for assessing immunotoxicity and is a much easier and cheaper tissue that can be sampled repeatedly from the same individual. This approach allows a refinement of sampling methodologies of experimental designs for toxicology and ecotoxicology and also reduces the number of animals required because the same individuals can be used for multiple comparisons (repeated measurements).10 In the case of genomics, the use of blood will eliminate fish exposure history differences as well as public concern about animal welfare and their use in experimental designs. The application of this approach could avoid the necessity of euthanizing animals to assess toxicant effects on target organs, which is a major drawback of these applications with regards to monitoring wildlife in ecotoxicology studies, particularly for endangered species. Fully fluorinated alkyls, also known as per and polyfluoro alkyl substances (PFAS), are considered one of the most ubiquitous types of endocrine-disrupting compounds (EDCs).11 Their wide use, high persistence, and bioaccumulative properties have led to an omnipresent occurrence in the environment. In particular, the presence of PFAS in the aquatic environment has been commonly documented.12,13 The accumulation of PFAS has been widely detected in freshwater and marine fish,14 with the liver and blood serving as the main body compartments for the bioaccumulation of PFAS in fish including the fathead minnow.15,16 Toxic effects of PFAS on biota have been studied for almost four decades in animals17 and are largely documented in the scientific literature.18−20 Reported health-related changes in fish range from general effects such as reduced growth and survival and reproductive organ impairment to the alteration of specific cellular pathways such as lipid metabolism, oxidative stress, DNA damage, immunotoxicity, and estrogenic effects.19 The induction of the peroxisome proliferator receptors (PPAR) seems to have a pivotal role in triggering several deleterious effects in mammals such as DNA damage, the production of reactive oxygen species (ROS), and the alteration of lipid metabolism.21 In some fish species, such as rainbow trout, exposure to PFAS increases the expression of the estrogen receptor (ER), a response related to carcinogenesis, more so than alterations of expression of PPAR isoforms. The effect of PFAS on the immune system of fish has also been previously observed,22 including effects on the pro-inflammatory cytokines (TNF-α, IL-1β, and IL-8), which have been highlighted as indicators of immune toxicity produced by perfluoroctanesulfonic acid (PFOS) exposure.23,24
Our hypothesis was that blood, an alternative tissue, would respond to low levels of exposure to PFAS. This response would be different from liver (which is the primary target tissue for toxicological studies) but as informative to assess toxicity of the aquatic environment using fish. We used a transcriptomics approach to investigate the differential expression of gene responses of liver and blood to study whole blood as a nonlethal sampling option.
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MATERIALS AND METHODS Exposure Water Preparation. A total of 4 different treatments were prepared for a static exposure of the fish (Table S1): a control (0 μg/L), 2 PFOS (perfluorooctanesulfonic acid, CAS: 2795-39-3) concentrations including high PFOS (25 μg/L) and low PFOS (0.5 μg/L), and a PFAS mix consisting of 7 types of PFAS at concentrations similar to those found previously downstream from a wastewater treatment plant25 to mimic the mixtures usually found in waters with wastewater influence. The mixture treatment contained the following 7 types of PFAS: PFBA (perfluorobutanoate, CAS: 375-22-4); PFHxA (perfluorohexanoic acid, CAS: 307-24-4); PFHpA (perfluoroheptanoic acid, CAS: 375-85-9); PFOA (perfluorooctanoic acid, CAS: 335-67-1); PFOS (perfluorooctanesulfonic acid, CAS: 2795-39-3); PFNA (perfluorononanoic acid, CAS: 375-95-1); and PFDA (perfluorodecanoic acid, CAS: 335-76-2). We added 50 mL of PFAS treatment preparations into a pre-cleaned fiberglass distribution cylinder containing 38 L of carbon-filtered and dechlorinated municipal water (Gainesville, FL) to give the desired final concentrations. For the control group, Milli-Q water and a carrier, triethylene glycol (TEG), were mixed in equivalent volumes for all treatments. All PFAS were purchased from Wellington Laboratories (Ontario, Canada) with a purity of over 99%. At the end of the exposures, 1 L of water samples were collected from 1 tank of each treatment in polypropylene bottles for the analytical chemistry analysis of 7 types of PFAS using U.S. Environmental Protection Agency (EPA) method no. 537.26 Water Analyses. To corroborate the concentrations used in the exposures, the seven types of PFAS were analyzed. The detection of PFAS was performed using a high-performance liquid chromatography system coupled with tandem mass spectrometry (HPLC/ESI-MS/MS), allowing for a limit of detection (LOD) of 1.0 ng/L, and a limit of quantification (LOQ) of 4 ng/L, as previously reported by RodriguezJorquera et al.25 Quality control and standards used were as previously reported.27,28 More details about water chemistry analysis are included in the Supporting Information. Fish Exposure and Tissue Collection. Reproductively mature pond-reared fathead minnows (FHM) were purchased from Andersen Minnow Farm (Lonoke, AR) approximately 6 months before the exposures. A total of 32 males were separated from the common tank 2 weeks before the experiment and placed in the treatment aquaria for 48 h. Each exposure was conducted in quadruplicate, and each aquarium contained two male FHM in 4 L of treatment water. All exposures were static. Positions of the treatment tanks were randomized, and test initiation times were staggered to ensure an exposure and sampling interval of 48 h. The fish were not fed during the experiment. The temperature range of the water was 24−26 °C, and a photoperiod of 16 h of light and 8 h of dark was used. At the conclusion of the exposures, fish were anesthetized with MS-222 and weighed to the nearest 0.1 g. B
DOI: 10.1021/acs.est.8b03603 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology Average fish weights were 3.68 g (control), 3.19 g (low PFOS, 0.5 μg/L), 3.99 g (high PFOS, 25 μg/L), and 3.47 g (PFAS mix). The testes were removed and preserved for histological analysis to confirm sex and sexual maturity stage. Liver tissues and whole blood were flash-frozen using liquid nitrogen and stored at −80 °C until RNA extraction. Samples of liver and blood (a minimum of 40 μL) from the same individuals (4 for each treatment) were used for RNA extraction and microarray processing (Figure 1). All procedures involving live fish were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee (IACUC).
publicly available EST sequences at the time plus genes that had been identified through suppressive subtractive hybridization in the laboratory and also contained 536 control probes. A total of four biological replicates were used for RNA isolation from FHM livers and blood using the same individuals to eliminate exposure history as a variable when comparing the two gene expression profiles. Microarray hybridizations were performed according to the Agilent “onecolor microarray” (document no. G4140-90040 version 6.5) using Cyanine 3 (Cy3) (Agilent, Palo Alto, CA). For blood and liver samples, 25 and 50 ng of total RNA per sample, respectively, were used to produce cDNA by reverse transcription using the poly(A) RNA present in the starting total RNA sample. Each sample contained a specific activity of >8.15 pmol Cy3/μg, and amounts were adjusted to a final mass of 600 ng per sample for hybridization. A final volume of 25 μL containing fragmented cRNA was added to the microarrays, and then hybridization proceeded for 17 h at 65 °C. Then, microarrays were washed according to the Agilent protocol and kept in the dark until scanning on an Agilent G2505B microarray scanner (same day). Data extraction was performed using Agilent Feature Extraction software (version 9.5). Bioinformatics. Raw expression data (gProcessedSignal) were imported into JMP Genomics v5 (SAS, Cary, NC) and log 2 transformed and normalized by LOESS before performing ANOVA to identify differentially regulated transcripts. Differentially regulated transcripts with a p value of
