BLOOD TRANSCRIPTOMICS ANALYSIS OF FISH EXPOSED TO

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Environmental Measurements Methods

BLOOD TRANSCRIPTOMICS ANALYSIS OF FISH EXPOSED TO PERFLUORO ALKYLS SUBSTANCES: ASSESSMENT OF A NON-DESTRUCTIVE SAMPLING TECHNIQUE FOR ADVANCING AQUATIC TOXICOLOGY RESEARCH. Ignacio Alejandro Rodriguez-Jorquera, Reyna Cristina Colli-Dula, Kevin J Kroll, B. Sumith Jayasinghe, Maria Virginia Parachu Marco, Cecilia Silva-Sanchez, Gurpal S. Toor, and Nancy D. Denslow Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b03603 • Publication Date (Web): 20 Dec 2018 Downloaded from http://pubs.acs.org on December 24, 2018

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Environmental Science & Technology

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BLOOD TRANSCRIPTOMICS ANALYSIS OF FISH EXPOSED TO PERFLUORO ALKYLS SUBSTANCES: ASSESSMENT OF A NON-LETHAL SAMPLING TECHNIQUE FOR ADVANCING AQUATIC TOXICOLOGY RESEARCH.

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Ignacio A. Rodríguez-Jorquera, 1,2,3* R. Cristina Colli-Dula, 3,4 Kevin Kroll, 3 B. Sumith Jayasinghe, 3 Maria V. Parachu Marco, 5,6,7 Cecilia Silva-Sanchez, 3 Gurpal S. Toor, 8 and Nancy D. Denslow 3

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1 Centro

de Humedales Río Cruces (CEHUM), Universidad Austral de Chile, Independencia 641,Valdivia, Región de los Ríos, Chile.

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2 Interdisciplinary

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3 Department

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7 Proyecto

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*Corresponding Author: [email protected]

Ecology Program, School of Natural Resources and Environment, Soil and Water Science Department, University of Florida, Gainesville, FL, USA. of Physiological Sciences & Center for Environmental and Human Toxicology, University of Florida, Gainesville, FL, USA. 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 s/n, CP 3000, Santa Fe, Argentina. 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, MD, USA.

ACS Paragon Plus Environment

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Abstract

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In contrast to mammals, the blood from other vertebrates such as fish contains nucleated red

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cells. Using a fathead minnow (Pimephales promelas) oligonucleotide microarray, we compared

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altered transcripts in the liver and whole blood after exposure to environmentally relevant

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concentrations of perfluorooctanesulfonic acid (PFOS) and a mixture of seven types of perfluoro

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alkyl substances (PFAS) including perflurooctanoic acid (PFOA). We used qPCR and cell based

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assays to confirm the main effects and found that blood responded with a greater number of

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altered genes than the liver. The exposure to PFAS altered similar genes with central roles in a

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cellular pathway in both tissues, including estrogen receptor alpha and peroxisome proliferator

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activator beta, indicating that the genes previously associated with PFAS exposures are

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differentially expressed in blood and liver. The altered transcripts are involved with cholesterol

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metabolism and mitochondrial function. Our data confirmed that PFAS are weak xenoestrogens

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and exert effects on DNA integrity. Gene expression profiling from blood samples not related

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with the immune system including very low-density lipoprotein, vitellogenin, estrogen receptor,

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and thyroid hormone receptor demonstrated that blood is a useful tissue to assess endocrine

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disruption in non-mammalian vertebrates. We conclude that the use of blood as non-lethal

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sampling in genomics studies is informative and particularly useful to assess effects of pollution

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in endangered species. Lastly, using blood will reduce animal use and widen the experimental

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design options to study effects of contaminants exposure on wildlife.

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Keywords: Perfluoro Alkyl Substances, Non-lethal Sampling, DNA damage, Xenoestrogens,

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Cholesterol Metabolism, Endocrine Disruption, Re-sampling.


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TABLE OF CONTENTS (TOC) ART

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INTRODUCTION Aquatic pollution affects biodiversity at local, regional, and global scales 1. In

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response to this issue, several techniques have been developed to research the impact of

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pollution on biota. Recently, genomic tools have improved our understanding of how

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chemicals impact the environment, revolutionizing both the fields of toxicology and

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ecotoxicology. 2 Specifically, transcriptomic analyses have been used to understand the

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effects of aquatic pollution in biota.3–5 Transcriptomics profiling allows researchers to

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perform analysis of thousands of genes at a time, to identify expression changes in response

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to chemical exposures, which can help to better understand the mode of action associated

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with biological responses to these exposures.2

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Despite the major advances in techniques in environmental toxicology and

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ecological risk assessment, improvements in data collection are necessary to understand the

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effects of pollution at the population level. For instance, the duration and consistency of

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pollutant effects, adaption and recovery periods collected in laboratory micro- and macro-

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mesocosm experiments should be evaluated over time to ensure a realistic response.6 In this

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context, the development of non-lethal techniques to assess pollution effects on the same

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individual will help to advance the understanding and action of pollution. Therefore, the

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use of blood emerges as an ideal tissue to assess changes in physiology in vertebrates

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caused by external stressors, particularly the non-mammalian vertebrates that have

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nucleated red cells and functional mitochondria.7

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Advances in genomics approaches, especially for dealing with specific issues

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affecting human subjects, allow the use of blood as a non-lethal sampling method to

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analyze gene expression signatures for toxicity determination.8,9 Moreover, blood is a


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logical choice for assessing immunotoxicity and is a much easier and cheaper tissue that

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can be sampled repeatedly from the same individual. This approach allows a refinement of

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sampling methodologies of experimental designs for toxicology/ecotoxicology and also

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reduces the number of animals required since the same individuals can be used for multiple

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comparisons (repeated measurements).10 In the case of genomics, the use of blood will

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eliminate fish exposure history differences as well as public concern about animal welfare

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and their use in experimental designs. The application of this approach could avoid the

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necessity to euthanize animals to assess toxicant effects on target organs, which is a major

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drawback of these applications with regards to monitoring wildlife in ecotoxicology

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studies, particularly for endangered species.

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Fully fluorinated alkyls, also known as per and polyfluoro alkyl substances (PFAS),

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are considered one of the most ubiquitous types of endocrine disrupting compounds

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(EDCs)11. Their wide use, high persistence and bioaccumulative properties have led to an

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omnipresent occurrence in the environment. Particularly, the presence of PFAS in the

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aquatic environment has been commonly documented.12,13 The accumulation of PFAS has

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been widely detected in freshwater and marine fish14 with the liver and blood serving as the

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main body compartments for the bioaccumulation of PFAS in fish including the fathead

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minnow.15,16 Toxic effects of PFAS on biota have been studied for almost four decades in

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animals17 and are largely documented in the scientific literature.18–20

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Reported health-related changes in fish range from general effects such as reduced

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growth, survival and reproductive organ impairment to alteration of specific cellular

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pathways like lipid metabolism, oxidative stress, DNA damage, immunotoxicity, and

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estrogenic effects.19 The induction of the peroxisome proliferator receptors (PPAR) seem to


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have a pivotal role in triggering several deleterious effects in mammals such as DNA

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damage, production of reactive oxygen species (ROS) and alteration of lipid metabolism.21

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In some fish species, such as rainbow trout, exposure to PFAS increases the expression of

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the estrogen receptor (ER), a response related with carcinogenesis, more so than alterations

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of expression of PPAR isoforms. The effect of PFAS on the immune system of fish has also

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been previously observed,22 including effects on the pro-inﬂammatory cytokines (TNF-α,

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IL-1β, and IL-8), which have been highlighted as indicators of immune-toxicity produced

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by perfluoroctane sulfonic acid (PFOS) exposure.23,24

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Our hypothesis was that blood, an alternative tissue, would respond to low levels of

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exposure to PFAS. This response would be different from liver (which is the primary

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target-tissue for toxicological studies) but as informative to assess toxicity of the aquatic

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environment using fish. We used a transcriptomics approach to investigate the differential

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expression of gene responses of liver and blood in order to study whole blood as a non-

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lethal sampling option.

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MATERIALS AND METHODS

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Exposure Water Preparation

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Four different treatments were prepared for a static exposure of the fish (Table S1,

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supporting information). A control (0 g/L); two PFOS (Perfluorooctanesulfonic acid,

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CAS: 2795-39-3) concentrations: PFOS High (25 g/L), PFOS Low (0.5 g/L); and a

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PFAS Mix, consisting of 7 types of PFAS at concentrations similar to those found

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previously downstream from a wastewater treatment plant 25 to mimic the mixtures usually

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found in waters with wastewater influence. The mixture treatment contained the following


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seven types of PFAS: PFBA (Perfluorobutanoate, CAS: 375-22-4); PFHxA

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(Perfluorohexanoic acid, CAS: 307-24-4); PFHpA (Perfluoroheptanoic acid, CAS: 375-85-

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9); PFOA (Perfluorooctanoic acid, CAS: 335-67-1); PFOS (Perfluorooctanesulfonic acid,

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CAS: 2795-39-3); PFNA (Perfluorononanoic acid, CAS: 375-95-1); PFDA

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(Perfluorodecanoic acid, CAS: 335-76-2). We added 50 mL PFAS treatment preparations

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into a pre-cleaned fiberglass distribution cylinder containing 38 L of carbon filtered and de-

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chlorinated municipal water (City of Gainesville, Florida, USA) to give the desired final

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concentrations. For the control group, Milli-Q water and a carrier Triethylene glycol (TEG)

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were mixed in equivalent volumes for all treatments. All PFAS were purchased from

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Wellington labs (Ontario, Canada) with purity over 99%. At the end of the exposures, 1 L

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of water samples were collected from one tank of each treatment in polypropylene bottles

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for analytical chemistry analysis of seven types of PFAS using EPA Method 537.26

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Water Analyses

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To corroborate the concentrations used in the exposures, the seven types of PFAS

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were analyzed. The detection of PFAS was performed using a high performance liquid

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chromatography system coupled with tandem mass spectrometry (HPLC/ESI-MS/MS)

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allowing for a limit of detection (LOD) of 1.0 ng/L, and a limit of quantification (LOQ) of

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4 ng/L, as previously reported by Rodriguez-Jorquera et al. 25 Quality control and standards

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used were as previously reported.27,28 More details about water chemistry analysis are

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included in the supplementary information section.

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Fish Exposure and Tissue Collection


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Reproductively mature pond-reared fathead minnow (FHM) were purchased from

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Andersen Minnow Farm (Lonoke, Arkansas) approximately 6-months before the

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exposures. Thirty-two males were separated from the common tank 2-weeks before the

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experiment and placed in the treatment aquaria for 48 h. Each exposure was conducted in

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quadruplicate and each aquarium contained two male FHM in 4 L of treatment water. All

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exposures were static. Positions of the treatment tanks were randomized and test initiation

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times were staggered to ensure an exposure/sampling interval of 48 h. The fish were not fed

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during the experiment. The temperature range of the water was 24–26 °C and the

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photoperiod of 16 h light: 8 h dark was used. At the conclusion of the exposures, fish were

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anesthetized with MS-222 and weighed to the nearest 0.1 g. Average fish weights were

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3.68 g (control); 3.19 g (PFOS Low 0.5 ug/L); 3.99 g (PFOS High 25 ug/L); and 3.47 g

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(PFAS Mix). The testes were removed and preserved for histological analysis to confirm

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sex and sexual maturity stage. Liver tissue and whole blood was flash frozen using liquid

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nitrogen and stored at -80 °C until RNA extraction. Samples of liver and blood (a minimum

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of 40 uL) from the same individuals (four for each treatment) were used for RNA

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extraction and microarray processing (Figure 1). All procedures involving live fish were

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reviewed and approved by the University of Florida Institutional Animal Care and Use

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Committee (IACUC).

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Microarray Analysis

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Total RNA was extracted following the RNA STAT-60 reagent protocol (Tel-Test,

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Friendswood, TX, USA). RNA was reconstituted in RNAsecure (Ambion; New York,

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USA), and DNase-treated with Turbo DNA-free (Ambion; New York, USA). In order to

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extract RNA from blood, column purification was used (RNeasy® Mini Kit Qiagen


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®;Limburg, Germany). The RNA quantity for microarray analysis was measured using the

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NanoDrop ND-1000 (Nanodrop Technologies, Wilmington, DE) and RNA quality was

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evaluated using the Agilent 2100 BioAnalyzer with the RNA 6000 Nanochip. RNA

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integrity numbers (RIN) ranges were 8.1 to 9.3 for blood samples and 7.5 to 9.4 for liver

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samples.

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An FHM 8 X 15K oligonucleotide microarray manufactured by Agilent (Palo Alto,

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CA) and designed in our laboratory (GEO: GPL9248) was used in this study.29 The array

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consisted of 15,744 oligonucleotide probes based on publicly available EST sequences at

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the time plus genes that had been identified through suppressive subtractive hybridization

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in the laboratory and also contained 536 control probes. Four biological replicates were

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used for RNA isolation from FHM livers and blood using the same individuals to eliminate

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exposure history as a variable when comparing the two gene expression profiles.

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Microarray hybridizations were performed according to the Agilent "One-color

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microarray”- (document no. G4140-90040 v6.5) using Cyanine 3 (Cy3) (Agilent, Palo Alto,

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CA). For blood and liver samples, 25 ng and 50 ng of total RNA per sample respectively

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were used to produce cDNA by reverse transcription using the poly (A) RNA present in the

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starting total RNA sample. Each sample contained a specific activity >8.15 pmol Cy3/ug,

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and amounts were adjusted to a final mass of 600 ng per sample for hybridization. A final

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volume of 25 μL containing fragmented cRNA was added to the microarrays, and then

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hybridization proceeded for 17 h at 65 ºC. Then, microarrays were washed according to the

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Agilent protocol and kept in the dark until scanning on an Agilent G2505B microarray

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scanner (same day). Data extraction was performed using Agilent Feature Extraction

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software (v9.5).


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Figure 1. General procedures of blood and liver gene expression analysis in fish using cDNA microarrays. In this study, the fish were euthanized.


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Bioinformatics

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Raw expression data (gProcessedSignal) were imported into JMP Genomics v5

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(SAS, Cary, NC) and log 2-transformed and normalized by LOESS before performing

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ANOVA to identify differentially regulated transcripts. Differentially regulated transcripts

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p < 0.01; fold change greater than ± 1.5 were subjected to hierarchical clustering. Distance

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calculations were performed with the program Cluster 3.0 30 using Euclidean distance as a

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similarity metric and average linkage as a clustering method and visualized using the Java

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Tree View software.31

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To evaluate gene expression changes through pathway analysis, human orthologs

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were found for FHM genes. Gene Ontology (GO) analysis was used to determine the

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biological processes containing over represented genes using Fisher Exact Test (p

BLOOD TRANSCRIPTOMICS ANALYSIS OF FISH EXPOSED TO

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