Establishment, Immortalization and Toxicological Applications of Primary Skin Fibroblasts from Three Sea Turtle Species

Home , Flatback sea turtle

Sarah J. Webb, B.S., M.S.

A DISSERTATION

ENVIRONMENTAL TOXICOLOGY

Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for The Degree of

DOCTOR OF PHILOSOPHY

Approved

Dr. Céline A. J. Godard-Codding (Chair of the Committee)

Dr. Christine A. Bishop

Dr. Michael J. Hooper

Dr. Christopher J. Salice

Dr. Kamaleshwar P. Singh

Dr. Mark Sheridan Dean of the Graduate School

December, 2014

Texas Tech University, Sarah J. Webb, December 2014

Acknowledgements

This work was only possible through contributions of the time and knowledge of many people, without whom I would never have been able to complete my doctoral studies. Dr. Céline Godard-Codding has been a supportive and encouraging mentor who has provided me with excellent guidance throughout the formation and completion of my work. The opportunity to work with endangered species is rare, and I appreciate greatly the chance I had in her laboratory at Texas Tech. I am also deeply grateful for the experiences she provided, including travel to scientific conferences and to do hands on work with real, live sea turtles. I would also like to thank my doctoral committee: Dr. Christine Bishop, Dr. Michael Hooper, Dr. Christopher Salice, and Dr. Kamaleshwar Singh have all been extremely helpful by answering any questions I may have. Dr. Singh in particular has answered many of my cell culture questions as I refined my techniques and deepened my knowledge of cell culture theory and practice.

In addition to my committee, many of the faculty at the Institute of Environmental and Human Health (TIEHH) have provided valuable insight and support. I especially appreciate Dr. Todd Anderson, Dr. Jaclyn Cañas-Carrell, Dr. Weimin Gao, Dr. Jonathan Maul, and Dr. Greg Mayer, all of whom I have gone to with questions at some point. They graciously answered my many questions thoroughly and without complaint when I appeared at their office doors with random questions. In addition to the faculty, the staff at TIEHH has been extremely helpful and I greatly appreciate their help with paperwork, etc. over the years. Stephanie White, Allyson Smith, Karinne Truelock, Stephanie Presley, and Stephanie Franco Alexander all provided invaluable assistance.

I would also like to thank our collaborators for their hard work and advice on the various aspects of my project. Without Benjamin Higgins and Lyndsey Howell at the NOAA/NMFS sea turtle facility in Galveston, Texas my work would not have been possible. I thoroughly enjoyed the time Ben spent teaching me about the husbandry of sea turtles in captivity, and appreciate all of the times he answered questions for me about sea turtles. I greatly appreciate Kathryn Furr at the Texas Tech Health Sciences Center, who was instrumental in getting flow cytometry data and kindly devoted several days of her time to analyzing my data.

Texas Tech University, Sarah J. Webb, December 2014

Dr. Patricia McClellan-Green was a role model to me, as a toxicologist who had worked on sea turtles during her career as well as being a mother who had successfully raised children while maintaining a career in science. She was kind enough to open her home to me for a week while I worked with sea turtle hatchlings in North Carolina, and I thoroughly enjoyed the time I got to spend visiting with her and talking about her work.

Without the support of my labmates I would not have been able to complete my work. I would especially like to thank those students who trained me in the lab, Jennifer Cole, Sandy Wiggins, and Greg Zychowski. Becca Pezdek is amazing and awesome and I’m so glad we were able to work together. Jan Yacabucci and I spent many enjoyable hours sitting in the dark working with samples. Subsequent lab members have provided invaluable help in the laboratory and company along the way: Kayla Campasino, Lauren McGeorge, Micah Thal, Kimberly Smelker, Andrew East, and Mary Gendron. Best wishes to our new lab members, Jocylin Pierro and Courtney Alexander as they begin their graduate work at Texas Tech.

The friends I have made at Texas Tech have been wonderful, and without them I never would have survived. Darcy Chase, Kristyn Urban, Kim Wooten, Kaylyn Germ, and Anna Hoffarth are some of the best friends I have had and I’m so glad I had the opportunity to meet them while in Lubbock. They provided excellent advice and comic relief throughout my time here and kept my spirits up along the way. I appreciate the food, drink, babysitting, and company they provided over the years. Long live WW!

I never would have been able to complete my graduate studies without the help and support of my family. My very best friends, Kara Allen and Amanda White have helped to keep me sane. My parents and sister have been incredibly supportive over the many years I have spent on graduate studies. I’d like to thank my husband, Ryan Webb, who has supported my efforts to earn my degrees. He has been amazing: cooking, cleaning and caring for our daughter and essentially being a single parent many nights. Last, I dedicate all of my work to my daughter, Lilah Webb. I love you more than anything.

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Table of Contents

Acknowledgements ...... ii Abstract ...... v List of Tables ...... vii List of Figures ...... ix Chapter 1 ...... 1 1.1 Introduction and Historical Perspective ...... 1 1.2 Culture Methods ...... 5 1.3 Suggestions for Successful Culture of Chelonian Cells ...... 7 1.4 References ...... 14 Chapter 2 ...... 19 2.1 Introduction ...... 19 2.2 Experimental Methods ...... 22 2.3 Results ...... 28 2.4 Discussion ...... 29 2.6 References ...... 44 Chapter 3 ...... 53 3.1 Introduction ...... 53 3.2 Methods ...... 55 3.3 Results ...... 56 3.4 Discussion ...... 56 3.4 References ...... 61 Chapter 4 ...... 65 4.1 Introduction ...... 65 4.2 Methods ...... 68 4.3 Results ...... 72 4.4 Discussion ...... 75 4.5 References ...... 100 Chapter 5 ...... 111 5.1 Introduction ...... 111 5.2 Methods ...... 113 5.3 Results ...... 115 5.4 Discussion ...... 115 5.4 References ...... 121

Texas Tech University, Sarah J. Webb, December 2014

Abstract

Pollution is a well-known threat to sea turtles but its impact is poorly understood. In vitro toxicity testing presents a promising avenue to assess and monitor the effects of environmental pollutants in these animals within the legal constraints of their endangered status. Reptilian cell cultures are rare and, in sea turtles, largely derived from animals affected by tumors. Here we describe the full characterization of primary skin fibroblast cell cultures derived from biopsies of multiple healthy loggerhead sea turtles (Caretta caretta), and the subsequent optimization of traditional in vitro toxicity assays to reptilian cells. Primary skin fibroblast cell cultures were also established and described for the green sea turtle (Chelonia mydas) and the Kemp’s ridley sea turtle (Lepidochelys kempii). Characterization of loggerhead sea turtle fibroblasts included validating fibroblast cells by morphology and immunocytochemistry, and optimizing culture conditions using growth curve assays with a fractional factorial experimental design. Two cell viability assays, two assays assessing sublethal cytotoxic effects, and two CYP1A biomarker assays were optimized in the characterized loggerhead sea turtle cells. MTT and lactate dehydrogenase (LDH) cell cycle analysis successfully showed cytotoxicity when cells were exposed to environmentally relevant concentrations of perfluorinated compounds. Using c flow cytometry, we detected no alterations to the cell cycle as a result of exposure to any compound tested in preliminary studies. Reactive oxygen species (ROS) assays showed significant inhibition of ROS production in cells exposed to phenanthrene. Cytochrome P4501A (CYP1A) expression was analyzed by quantitative PCR which detectedinduction of CYP1A5 in cells exposed to benzo(a)pyrene for 72 hours. Enzymatic activity was quantified via AROD analysis and showed BROD and PROD activity when cells were exposed to benzo(a)pyrene, PCB77, and PCB126, but no MROD or EROD activity. This research demonstrates validity of in vitro toxicity testing in sea turtles and highlights the need to optimize mammalian assays to reptilian cells. Additionally, immortalization of fibroblast cell lines derived from multiple, healthy sea turtles was attempted by the use of an hTERT insert transducted into the genome via a lentiviral vector. Transduction was unsuccessful for loggerhead and green

Texas Tech University, Sarah J. Webb, December 2014 sea turtle fibroblasts, but one line from a green sea turtle was successfully transduced. Immortal cells from the green sea turtle maintained normal fibroblastic morphology in culture when compared to primary cells from the same animal, but exhibited more rapid proliferation.

Texas Tech University, Sarah J. Webb, December 2014

List of Tables

Table 1.1. Summary of cell types and/or sources for cell cultures established from turtle species...... 9

Table 1.2. Summary of published cell culture establishment methods in turtles. Disaggregation refers to either enzymatic and/or physical breakdown of connective tissue. Explant refers to mincing tissue into small pieces used sterile forceps or scalpels...... 10

Table 1.3. Summary of published medium used for culture of turtle cells. RPMI 1640, Roswell Park Memorial Medium 1640; MEM, Minimum Essential Medium; DMEM, Dulbecco's Modified Eagle's medium; DMEM/F12, Dulbecco's Modified Eagle's medium: Nutrient Mixture F12; BME, Basal Medium Eagle...... 11

Table 1.4. Summary of published serum concentrations used in the culture of turtle cells...... 12

Table 1.5. Summary of published reported incubation temperatures for turtle cell cultures. If a variety of temperatures was tested, only the optimal temperature(s) was included...... 13

Table 2.1. Combinations tested by One Variable at a Time (OVAT) approach. Optimal conditions were determined based on growth as well as statistical differences in final cell counts at each time point (p≤0.05)...... 34

Table S1. Comparison of explant methods, including time to first passage and number of cells yielded at first passage...... 42

(3-1) Table S2. Fractional factorial design, 2III , generated by JMP ® 8.0. Matrix expressed in +/- format, and matrix expressed with designations for each factor/level (medium, % serum, surface). Temperature was not included as a factor since 30°C was selected as the optimal temperature prior to the FF design. The last row corresponds to the only combination not already tested by OVAT approach...... 43

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Table 4.1. Polycyclic aromatic hydrocarbon concentrations reported in the literature in marine turtle blood and/or tissue, in seawater, and in other marine species on which dose ranges were based...... 83

Table 4.2. Polychlorinated biphenyl concentrations reported in the literature in marine turtle blood and/or tissue, in other marine species, and used in vitro in other taxa on which dose ranges were based...... 84

Table 4.3. Perfluorinated compound concentrations reported in the literature in marine turtle blood and/or tissue, in seawater, in other marine species, and used in vitro in other taxa on which dose ranges were based...... 85

Table 5.1. Animals from which primary skin fibroblasts were exposed to hTERT lentivirus carrier in medium. Cells from the animal in bold were successfully transfected...... 86

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List of Figures

Figure 2.1. Growth curve characterization assays for OVAT analysis in Caretta caretta primary skin fibroblasts: A) Medium tests, B) Serum concentration tests, C) Temperature variability analysis, D) Coating/substrate analysis. Assays were performed in triplicate using cells from 3 or more animals. Bars represent standard error of the mean. Asterisks (*) indicate statistical significance at p≤0.05...... 35

Figure 2.2. Caretta caretta primary skin fibroblasts. A) Fibroblasts proliferating from an explant (100X, scale bar = 20μm); B) Fibroblasts in cell culture (100X, scale bar = 10μm); C) Vimentin labeling by immunocytochemistry in Caretta caretta primary skin fibroblasts (100X, scale bar = 50μm). Vimentin is visualized using a rhodamine- conjugated, goat-anti-rabbit IgG Red-X (red), and DAPI counterstained nuclei (blue). Images were captured by deconvolution fluorescence microscopy using a Hamamatsu Orca – ER high-speed camera on an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan) at 100X, and analyzed using Simple PCI software...... 36

Figure 2.3. Analysis of chromosomes from 24 cells from a loggerhead sea turtle. A) Karyotype of C. caretta primary fibroblasts showing 2n=56, with 24 macrochromosomes and 32 microchromosomes. B) Metaphase spread from a C. caretta fibroblast...... 37

Figure 2.4. PFOA cytotoxicity in Caretta caretta primary skin fibroblasts. A) MTT assays following 72 and 96 hour exposure to PFOA with percent viability of treated cells normalized to performance of control (DMSO treated) cells (means ± SEM, n=6 animals). B) LDH assays following 72 and 96 hour exposure to PFOA (means ± SEM, n=5 animals). Asterisks (*) indicate statistical significance at p≤0.05...... 38

Figure 2.5. Relative mRNA expression of CYP1A5 fold change (normalized to the 18S gene) in loggerhead sea turtle primary skin fibroblasts exposed to B(a)P for 72h (means ± SEM, n=6 animals). Different letters indicate statistically different means within different treatment groups (p≤0.05)...... 39

Figure 3.1. (A) Fibroblasts proliferating from a Kemp’s ridley sea turtle tissue biopsy 15 days following explant establishment. (B) Fibroblasts proliferating from a green sea turtle

Texas Tech University, Sarah J. Webb, December 2014 tissue biopsy 9 days following explant establishment. Images were captured by deconvolution fluorescence microscopy using a QImaging Go-3 high-resolution digital color microscope camera (100X, scale bar = 20μm)...... 58

Figure 3.2. (A) Primary fibroblasts from a Kemp’s ridley tissue biopsy 21 days following explant establishment. (100X, scale bar = 10μm) (B) Primary fibroblasts from a green sea turtle tissue biopsy 16 days following explant establishment (100X, scale bar = 20μm). Images were captured using a QImaging Go-3 high-resolution digital color microscope camera...... 59

Figure 3.3. (A) Fibroblasts from a Kemp’s ridley sea turtle tissue biopsy exhibiting parallel arrangement at ~90% confluence. (B) Fibroblasts from a green sea turtle tissue biopsy at ~90% confluence. Image was captured by deconvolution fluorescence microscopy using a QImaging Go-3 high-resolution digital color microscope camera (100X, scale bar = 20μm)...... 60

Figure 4.1. MTT assays assessing benzo(a)pyrene cytotoxicity in Caretta caretta primary skin fibroblasts (means + SEM; n=3 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, no significant effects. C) 72 hour exposure, significant effects occurring at the 10μM dose. D) 96 hour exposure, no significant effects. Asterisks (**) indicate statistical significance as compared to control (p≤0.01)...... 83

Figure 4.2. MTT assays assessing phenanthrene cytotoxicity in Caretta caretta primary skin fibroblasts (means +SEM; n=4 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, no significant effects. C) 72 hour exposure, no significant effects...... 84

Figure 4.3. MTT assays assessing naphthalene cytotoxicity in Caretta caretta primary skin fibroblasts, (means +SEM; n=4 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, no significant effects. C) 72 hour exposure, no significant effects...... 85

Texas Tech University, Sarah J. Webb, December 2014

Figure 4.4. MTT assays assessing PCB 77 cytotoxicity in Caretta caretta primary skin fibroblasts (means +SEM; n=5 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, no significant effects. C) 72 hour exposure, significant effects occurring at the 10 μM dose. D) 96 hour exposure, no significant effects. Asterisk (*) indicates statistical significance (p≤0.05)...... 86

Figure 4.5. MTT assays assessing PCB 126 cytotoxicity in Caretta caretta primary skin fibroblasts (means + SEM; n=5 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, no significant effects. C) 72 hour exposure, no significant effects. D) 96 hour exposure, no significant effects...... 87

Figure 4.6. MTT assays assessing perfluorooctanoic acid cytotoxicity in Caretta caretta primary skin fibroblasts (means + SEM; n=5 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, no significant effects. C) 72 hour exposure, significant effects occurring at the 500 μM dose. D) 96 hour exposure, significant toxicity occurring at the 500 μM dose. Asterisks (***) indicate statistical significance (p<0.001)...... 88

Figure 4.7. MTT assays assessing perfluorooctane sulfonate cytotoxicity in Caretta caretta primary skin fibroblasts (means +SEM; n=5 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, significant effects occurring at the 200 μM dose. B) 48 hour exposure, no significant effects. C) 72 hour exposure, significant effects occurring at the 200 μM dose. D) 96 hour exposure, no significant effects. Asterisks (*) indicate statistical significance (* p≤0.05, ** p≤0.01)...... 89

Figure 4.8. LDH assays assessing benzo(a)pyrene cytotoxicity in Caretta caretta primary skin fibroblasts (means ± standard deviation, n=5 animals, cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, significant effetcs occurring at the 10μM dose. C) 72 hour exposure, no significant effects. D) 96 hour exposure, significant effects occurring at the 10μM dose. Asterisk (*) indicates statistical significance (p≤0.05)...... 90

Texas Tech University, Sarah J. Webb, December 2014

Figure 4.9. LDH assays assessing PCB 77 cytotoxicity in Caretta caretta primary skin fibroblasts (means + SEM). A) 24 hour exposure (n=5 animals, cells dosed in triplicate), no significant effects. B) 48 hour exposure (n=4 animals, cells dosed in triplicate), no significant effects...... 91

Figure 4.10. LDH assays assessing PFOA cytotoxicity in Caretta caretta primary skin fibroblasts (means +SEM; n=5 animals; cells dosed in triplicate for each timepoint). A) 24 hour exposure, no significant effects. B) 48 hour exposure, significant toxicity occurring at the 500 μM dose. C) 72 hour exposure, significant effects occurring at the 500 μM dose. D) 96 hour exposure, significant effects occurring at the 500 μM dose. Asterisks (*) indicate statistical significance (** p≤0.01, *** p≤0.001)...... 92

Figure 4.11. ROS production in C. caretta cells following exposure to phenanthrene (n=3 animals, cells dosed in triplicate for each timepoint) A) with positive control shown, and B) without positive control. Significant reduction in ROS production occurred for every dose relative to control at every timepoint (p≤0.05), with the exception of 0.001 μM at 30 minutes and 1 hour...... 93

Figure 4.12. ROS production in C. caretta cells following exposure to phenanthrene (means +SEM; n=3 animals; cells dosed in triplicate) for one hour. A significant decrease in ROS was detected at every dose relative to control. Asterisks (*) indicate statistical significance ( *** p≤0.001)...... 94

Figure 4.13. Flow cytometry data (propidium iodide stain) showing cell cycle distribution of cells dosed for 72 hours with phenanthrene. No significant changes in the cell cycle were measured, based on S and G2/M phases (n=2 animals)...... 95

Figure 4.14. AROD assay following exposure to PCB77 for 72 hours in Caretta caretta primary skin fibroblasts, showing PROD activity (means +SEM; n=2 animals). No MROD, EROD, or BROD activity was detected...... 96

Figure 4.15. AROD assay following exposure to PCB126 for 72 hours in Caretta caretta primary skin fibroblasts, showing A) PROD activity (n=1 animal), and B) BROD activity (n=1 animal). No MROD or EROD activity was detected...... 97

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Figure 4.16. AROD assay following exposure to benzo(a)pyrene in Caretta caretta primary skin fibroblasts, showing BROD activity (means +SEM; n=3 animals)...... 98

Figure 4.17. AROD assay following exposure to benzo(a)pyrene in Caretta caretta primary skin fibroblasts, showing PROD activity (means +SEM; n=3 animals)...... 99

Figure 5.1. Immortalized cells from a green sea turtle, exhibiting the spindle morphology characteristic of fibroblasts in culture. Image was taken using a QImaging Go-3 high- resolution digital color microscope camera (100X, scale bar = 10μm)...... 118

Figure 5.2. Immortalized cells from a green sea turtle, approaching confluence. Cells did not form parallel arrangement when approaching confluence. Image was taken using a QImaging Go-3 high-resolution digital color microscope camera (100X, scale bar = 20μm)...... 119

Figure 5.3. The turtle from which cells were successfully immortalized, a stranded and rehabilitated wild green sea turtle (Chelonia mydas)...... 120

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Chapter 1

A Review of Cell Culture Methodologies and Applications in Chelonian Species

1.1 Introduction and Historical Perspective Animal cells have been cultured in a laboratory setting since Dr. Ross Harrison published the first paper on the successful culturing of cells in vitro at the beginning of the 20th century (Harrison 1907). Since then, improvements in culturing techniques and microscopy as well as the development of antibiotics to aid in the prevention of contamination have led to the widespread use of cell culture to study cellular anatomy and physiology, pharmaceuticals, disease, toxicity, cancers, virology, and more. While cells have been cultured from bacteria, plants, fungi, invertebrates, and vertebrates, most animal cell research is conducted using mammalian cells, although there are a few insect and fish cells lines that are regularly used in research (Freshney 2005). This review examines past work on chelonian cell culture including details on species used and culture conditions. We include an overview of culture establishment methodology, medium type and serum concentration, and incubation temperature. This work extensively and carefully reviewed the available literature on chelonian cell cultures, and any omission of published research is inadvertent. This review does exclude chelonian blood cell culture, as these cultures tend to be short-lived (1-2 weeks), require exposure to a mitogen for division and are not intended to produce cells for research via proliferation. Turtle species are rapidly disappearing, with 51% of turtle species listed as threatened and 22% listed as near threatened, for a total of 73% of turtle species in danger of extinction (Böhm, Collen et al. 2013). Threats to tortoises and freshwater turtle populations differ somewhat from threats to marine turtles. The biggest threats to tortoises and freshwater turtles include habitat destruction and collection for human consumption, the pet trade, and traditional medicines (Gibbons, Scott et al. 2000, Rhodin, Walde et al. 2011). Threats to marine turtle species are more convoluted and include fishing and shrimping activity, habitat destruction, boat strikes, and exposure to

Texas Tech University, Sarah J. Webb, December 2014 potentially toxic contaminants (Gibbons, Scott et al. 2000, Hays 2008, Bolten, Crowder et al. 2010). In order to fully understand threats such as disease and exposure to contaminants, it is essential to understand the physiology of these animals. Most traditional methods of study involve the euthanasia of animals and subsequent harvesting of tissues and organs. This use of animals is not usually a viable option when studying endangered species. Methods must be minimally invasive and non-lethal in order to understand the animals while avoiding disruption and depletion of a population in which every individual may be vital to the survival of the species. The use of cell cultures and in vitro research is an ideal option for the study of turtles, as cell cultures can be obtained from skin biopsies with little to no lasting impact on individual animals (Bjorndal, Reich et al. 2010). Additionally, other cell types can be obtained opportunistically from the tissues and organs of individual animals that are compromised due to injury or disease and do not qualify for rehabilitation. To date, cell culture has been described in only twelve of the almost 300 species of extant turtles, including five of the seven marine turtle species. This includes both immortal cell cultures, which have been altered to avoid senescence, and primary cell cultures, which have a finite lifespan before they reach senescence and die-off.

Tortoises and Freshwater Species In non-marine turtle species, cell culture has been described in peer-reviewed literature in 4 families: Emydidae, Podocnemididae, Testudinidae, and Trionychidae. The first species of turtle for which cell culture was described was the painted turtle, Chrysemys picta, in 1960 (Wolf, Quimby et al. 1960). Wolf and colleagues described methods of culturing cells from cold-blooded animals, including trout (Salmo gairdneri), a bullfrog (Rana catesbeiana), and ovarian cells from one painted turtle. No further work was done with C. picta until the 1980s, when five papers were published describing the culture of brain (Callard, Petro et al. 1980, Callard 1981) and thyroid (Chow, Yen-Chow et al. 1985, Chow, Yen-Chow et al. 1986, Chow, Yen-Chow et al. 1987) cells, which were used in physiological studies. Researchers in the 1960s continued work with chelonian cells with cultures established from the Greek tortoise (Testudo graeca) (Shindarov 1961, Fauconnier 1963,

Texas Tech University, Sarah J. Webb, December 2014

Fauconnier 1963, Falcoff and Fauconnier 1965, Clark, Cohen et al. 1970) and the eastern box turtle (Terrapene carolina) (Clark and Karzon 1967, Huang and Clark 1967, Clark, Cohen et al. 1970). From the Greek tortoise, kidney cells were applied to the investigation of multiple viruses in four separate studies (Shindarov 1961, Fauconnier 1963, Fauconnier 1963, Falcoff and Fauconnier 1965). An immortal cell line, the first for chelonians, was established from one eastern box turtle heart and designated TH-1 (terrapene heart) in 1967 (Clark and Karzon 1967). Heart cells were again isolated from eastern box turtles in another lab during the same year, along with cells derived from the spleen and lung, and were used for chromosome analysis (Huang and Clark 1967). Huang and Clark (1969) isolated primary and immortal cultures derived from the heart and lung of two additional species, a yellow spotted Amazon river turtle (Podocnemis unifilis), and a South American river turtle (P. expansa) (Huang and Clark 1969). Following the increasing use of chelonian cell culture for physiological research in the 1960s, Clark and colleagues published a paper in 1970 with the purpose of characterizing reptilian cell lines and with an emphasis on incubation temperature (Clark, Cohen et al. 1970). They attempted the establishment of cells from five species: the Greek tortoise, the eastern box turtle, a yellow spotted Amazon river turtle (P. unifilis), a musk turtle (Kinosternon subrubrum), and a snapping turtle (Chelydra serpentina) (Clark, Cohen et al. 1970). From these, they had success with yellow-spotted Amazon river turtle heart cells, cells from Greek tortoise spleen, and heart, lung, kidney, and spleen cells from the eastern box turtle. Establishment of cultures from the musk and snapping turtles was unsuccessful. In 2000, cell cultures were described for the Chinese soft-shell turtle (Pelodiscus sinensis, previously known as Trionyx sinensis) (Guo 2000, Li and Zhang 2000). The Chinese soft-shell turtle is extensively farmed for food in China, Japan, and Taiwan, and problems with disease have led to increased cell culture studies in recent years to provide a model for understanding red-neck disease (Fu, Luo et al. 2013). Both primary and immortal cultures have been established from this species, including cells derived from the spleen, heart, liver, kidney, skin, shell, smooth muscle cells from the carotid artery, and from whole embryos (Guo 2000, Li and Zhang 2000, Li, Zeng et al. 2010, Liu, Wang et al. 2012, Fu, Luo et al. 2013). Neuronal cell cultures were established from the red-

Texas Tech University, Sarah J. Webb, December 2014 eared slider (Trachemys scripta) in 2007. These primary cultures were derived from the brain, and were used in two studies to assess the anoxic conditions and reactive oxygen species production (Milton, Nayak et al. 2007, Nayak, Prentice et al. 2009). A summary of cell types established from turtles can be found in Table 1.1.

Marine Species The first paper published on marine turtle cell culture was in 1965, and reported on cells derived from the kidney of a green sea turtle (Chelonia mydas) with fibropapillomatosis (Waddell and Sigel 1965). These cultures were used for a variety of endpoints, including the study of viruses, determining optimal temperature for growth of reptilian cells, and obtaining a karyotype for the green turtle. A year later, cells were established from green sea turtle embryonic tissues, in a study that also established culture methods for three other reptiles, the Cunningham’s skink (Egernia cunninghami), the mountain dragon (Amphibolurus diemensis), and the leopard ctenotus (Sphenomorphus ocelliferum), and compared them to cells from chickens, mice, and the striped marsh frog (Limnodynastes peroni)(Stephenson 1966). This work confirmed that mammalian medium was acceptable for reptilian cells, and reported on the effects of different temperatures on reptilian cell growth. Cells were again cultivated from green sea turtles in the 1980s: an epithelial culture derived from the skin of a hatchling (Koment and Haines 1982) and two skin fibroblast cultures derived from adult green turtles with fibropapillomatosis (Mansell, Jacobson et al. 1989). Following this initial research into marine turtle cell culture, cells from the green sea turtle were used in at least eight more studies involving both primary and immortal cells derived from FP-positive animals which predominantly centered on fibropapillomatosis and toxicological research (Moore, Work et al. 1997, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999, Lu, Aguirre et al. 2000, Herbst, Chakrabarti et al. 2001, Work, Dagenais et al. 2009, Tan, Wang et al. 2010, Wang, Tong et al. 2013). Cell cultures from other marine turtle species were not described until 2012, when primary fibroblast skin cells were established from hawksbill sea turtles (Eretmochelys imbricata) (Fukuda, Kurita et al. 2012). The next year, cell cultures were cultured from hawksbill sea turtle embryos and used to test heat shock protein expression with

Texas Tech University, Sarah J. Webb, December 2014 increasing cell incubation temperature (Takeshita, Matsuda et al. 2013). In 2014, hawksbill cell cultures were used to test the cytotoxicity of heavy metals using cells from one animal (Wise, Xie et al. 2014). In 2014, the culture of loggerhead sea turtle primary fibroblasts was described in detail (Webb et al., Chapter 2). These cells were the first marine turtle cells for which growth conditions were fully characterized, including growth medium, growth temperature, medium serum content, growth substrate, and establishment method. In the same year a brief letter to the editor described the culturing of primary olive ridley (Lepidochelys olivacea) sea turtle cells (Fukuda, Katayama et al. 2014). Additionally, the establishment of primary cultures from Kemp’s ridley (Lepidochelys kempii) and green sea turtles has been described (Webb et al., Chapter 3. A summary of cell types established from marine turtles can be found in Table 1.1.

1.2 Culture Methods Method of Establishment All chelonian cell cultures, both primary and immortal, have been established by one of two methods: tissue disaggregation or tissue explant. Tissue disaggregation involves the use of one or more enzymes and/or physical methods such as passing through a screen or syringe to break down connective tissue in a sample and release living cells into solution. The tissue explant method of establishment involves cutting tissue into small pieces using sterile scissors or scalpel blade to allow the proliferation of cells from the tissue pieces. The majority of work establishing turtle cells, regardless of species, used tissue disaggregation as a method of establishment of cultures (Table 1.2). Three studies used both methods: Huang et al. in 1967 with eastern box turtle tissue, and Koment et al. in 1982 and Mansell et al. in 1989 with green sea turtle tissues. Both green sea turtle studies noted that the tissue explant method was more successful than tissue disaggregation (Koment and Haines 1982, Mansell, Jacobson et al. 1989).

Medium Type and Serum Concentration

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A variety of different medium options have been successfully used in sea turtle culture, including Roswell Park Memorial Medium 1640 (RPMI 1640), Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's medium (DMEM), DMEM/Nutrient Mixture F12 (DMEM/F12), Basal Medium Eagle (BME), Medium 199, Coon's modified Ham F12, Leibowitz, Paul's salt solution, TC199, and L15 medium. RPMI 1640 or MEM/DMEM were the most commonly used (50% of the studies, Table 1.3). In addition to choice of growth medium, the use of serum (usually bovine) has often proved essential in turtle cell culture. While bovine was the most common serum type, two early studies experimented with turtle or tortoise serum (Shindarov 1961, Fauconnier 1963). However, both studies recommended using mammalian serum such as fetal bovine serum (FBS) or bovine calf serum (BCS) due to its success in aiding cell proliferation and ease of access compared to reptilian serum. A range of 5-20% serum has been used in turtle cell cultures. Several studies optimized medium serum content, and of these two chose 10% (Wolf, Quimby et al. 1960, Lu, Nerurkar et al. 1999), one 5% (Koment and Haines 1982), and one 20% (Fu, Luo et al. 2013) as optimal concentrations. Over two thirds of described culture methods used 10% serum concentrations (Table 1.4). Temperature of Incubation The ideal incubation temperature has been tested repeatedly for turtle cells (Table 4). While incubation temperatures are consistently under the normal temperature for mammalian cultures (37°C), there have been a wide range of successful temperatures reported. Optimal incubation temperatures were tested for Greek tortoise cells in 1963, ranging from 22°C to 37°C. The study reported that cells proliferated more rapidly as temperature increased, with markedly decreased proliferation at any temperature below 24°C (Fauconnier 1963). Incubation temperatures were tested three separate times for the eastern box turtle. In 1967, Clark et al. established the TH-1 cell line and tested incubation temperatures of 23°C, 30°C, and 36°C, as well as storage of cells at 4°C and 14°C. They reported an optimal proliferation temperature of 23°C, unstable growth at 30°C, and also noted that cells remained viable indefinitely at 14°C (Clark and Karzon 1967). In the same year, Clark and Karzon further tested the effects of temperature on this cell line and found that while early passage cells were unable to proliferate at

Texas Tech University, Sarah J. Webb, December 2014 temperatures above 30°C, alterations occurred with repeated division that allowed cultures to proliferate at 36°C (Clark and Karzon 1967). Huang and Clark tested the effects of growth temperature on karyotype, and found that cell lines cultured at 30°C had more polyploidy and a more variable karyotype than those cultured at 23°C (Huang and Clark 1967). In 1970, Clark and colleagues reported that both 23°C and 30°C produced acceptable levels of box turtle cell proliferation in separately derived cell lines, although cells cultured at 30°C had less stable cell morphology as compared to 23°C (Clark, Cohen et al. 1970). Incubation temperatures were tested for cells from the Chinese soft- shell turtle in three different studies, and optimal temperatures of 25°C (Li and Zhang 2000), 28°C (Fu, Luo et al. 2013), and 30°C (Liu, Wang et al. 2012) were reported. Two other studies observed rapid cell proliferation at 37°C but associated with increased granulation and disruption of morphology (Fu, Luo et al. 2013) and increased cell death (Liu, Wang et al. 2012). Temperatures ranging from 19°C to 37.5°C were used for incubation of turtle cells among different studies, but most fell between 23°C and 30°C. A range of temperatures was tested for green sea turtle cells in 1966, and while cell proliferation was observed at 23-25°C, 37.5°C produced the best population growth (Stephenson 1966). This study also reported that cells remained viable with no proliferation at 3°C. In 1997, temperature was again tested for green sea turtle cells, and 30°C was reported as the optimal growth temperature, with decreased proliferation at 25°C (Moore, Work et al. 1997). Growth temperatures were also optimized for hawksbill (Takeshita, Matsuda et al. 2013) and loggerhead (Webb et al., Chapter 2) sea turtles. A range of temperatures from 22°C to 37°C was tested for the hawksbill, with 33°C reported as optimal (Takeshita, Matsuda et al. 2013). For the loggerhead, 25°C, 30°C, and 35°C were tested, with optimal proliferation at 30°C (Webb et al., Chapter 2). Overall, the most common incubation temperature used was 30°C (Table 1.5).

1.3 Suggestions for Successful Culture of Chelonian Cells This review of the literature provides a range of conditions that have proven optimal for the culturing of turtle cells. This information can be used as a starting point

Texas Tech University, Sarah J. Webb, December 2014 when attempting cultures on new chelonian species. There are several conditions that need to be considered and ideally optimized when starting new cultures. The first of these is establishment method. While tissue disaggregation was used more commonly overall, the tissue explant method has been successful for the species in which it was tested. There were not any specific advantages reported with either method. A variety of medium types have been used for chelonian cultures, with 50% of successful studies using RPMI 1640 or MEM/DMEM, and, thus, we recommend their use as a starting option. The use of FBS or BCS is standard with previously established turtle cell cultures, with 10% by far the most common concentration used. A wide range of incubation temperatures have been tested, with successful proliferation most often occurring between 23°C and 30°C. Because there is such wide variation in success for different species, we highly recommend optimizing incubation temperature for new cultures. In summary, we recommend using RPMI1640 medium or MEM/DMEM, 10% serum concentration, and incubation of cultures between 23°C and 30°C. To aid in reproducibility of experimental results, it is ideal to have standardized, optimized conditions available. We highly recommend that culture conditions be optimized, as culture conditions can have significant effects on the outcome of assays (Raz, Fogler et al. 1979, Hahn and Shiu 1983, Hestermann, Stegeman et al. 2002). Two papers have reported a thorough characterization of growth conditions on which this optimization could be modeled, Webb et al. (Chapter 2) for the loggerhead sea turtle and Fu et al. (2013) for the Chinese soft-shelled turtle. These studies tested medium type, serum content, and incubation temperature, reported on the normal karyotype of the cells, and positively identified the cell type using immunocytochemistry.

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Paper Species Cell Type Paper Species Cell Type Freshwater and Land Species Marine Species Wolf et al. 1960 Chrysemys picta ovarian cells Waddell et al. 1965 Chelonia mydas kidney Callard et al. 1980 Chrysemys picta Forebrain cells Moore et al. 1997 Chelonia mydas kidney Chow et al. 1985 Chrysemys picta thyroid follicular cells Lu et al. 1999 Chelonia mydas kidney Huang et al. 1967 Terrapene carolina heart Stephenson et al. 1966 Chelonia mydas embryonic cells Clark et al. 1967 Terrapene carolina heart Moore et al. 1997 Chelonia mydas liver Clark et al. 1970 Terrapene carolina kidney Moore et al. 1997 Chelonia mydas heart Clark et al. 1970 Terrapene carolina lung Lu et al. 1999 Chelonia mydas brain Clark et al. 1970 Terrapene carolina spleen Lu et al. 1999 Chelonia mydas lung Shindarov et al. 1962 Testuda graeca kidney Lu et al. 1999 Chelonia mydas spleen Fauconnier et al. 1963 Testuda graeca kidney Koment et al. 1982 Chelonia mydas skin epithelials Falcoff & Fouconnier 1965 Testuda graeca kidney Mansell et al. 1989 Chelonia mydas skin fibroblasts Clark et al. 1970 Testuda graeca spleen Herbst et al. 1998 Chelonia mydas skin fibroblasts Clark et al. 1970 Podocnemis unifilis heart Herbst et al. 2001 Chelonia mydas skin fibroblasts Huang et al. 1969 Podocnemis expansa spleen Work et al. 2009 Chelonia mydas skin fibroblasts Huang et al. 1969 Podocnemis expansa lung Moore et al. 1997 Chelonia mydas muscle Huang et al. 1969 Podocnemis expansa heart Takeshita et al. 2013 Eretmochelys imbricata muscle cells Milton et al. 2007 Trachemys scripta brain Fukuda et al. 2012 Eretmochelys imbricata skin fibroblasts Nayak et al. 2009 Trachemys scripta neuronal cells Wise et al. 2014 Eretmochelys imbricata skin fibroblasts Clark et al. 1970 Terrapene carolina heart Fukuda et al. 2014 Lepidochelys olivacea skin fibroblasts Li et al. 2000 Pelodiscus sinensis heart fibroblasts Webb et al. 2014 Caretta caretta skin fibroblasts Li et al. 2010 Pelodiscus sinensis heart

Fu et al. 2013 Pelodiscus sinensis arterial cells

Guo et al. 2000 Pelodiscus sinensis splenocyte

Li et al. 2010 Pelodiscus sinensis kidney

Li et al. 2010 Pelodiscus sinensis liver

Li et al. 2010 Pelodiscus sinensis spleen

Liu et al. 2012 Pelodiscus sinensis embryonic cells

Table 1.1. Summary of cell types and/or sources for cell cultures established from turtle species.

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Paper Species Culture Technique Wolf et al. 1960 Chrysemys picta Disaggregation Callard et al. 1980 Chrysemys picta Disaggregation Chow et al. 1985 Chrysemys picta Disaggregation Clark and Karzon 1967 Terrapene carolina Disaggregation Huang and Clark 1967 Terrapene carolina Explant Clark et al. 1970 Terrapene carolina Both, primarily explant Shindarov 1962 Testuda graeca Disaggregation Fauconnier 1963 Testuda graeca Disaggregation Falcoff et al. 1965 Testuda graeca Disaggregation Clark et al. 1970 Testuda graeca Both, primarily explant Huang and Clark 1969 Podocnemis unifilis Explant Clark et al. 1970 Podocnemis unifilis Both, primarily explant Huang and Clark 1969 Podocnemis expansa Explant Milton et al. 2007 Trachemys scripta Disaggregation Nayak et al. 2007 Trachemys scripta Disaggregation Li et al. 2010 Pelodiscus sinensis Disaggregation Liu et al. 2012 Pelodiscus sinensis Disaggregation Fu et al. 2013 Pelodiscus sinensis Explant Stephenson 1966 Chelonia mydas Disaggregation Both, explant more Koment et al. 1982 Chelonia mydas successful Both, explant more Mansell et al. 1989 Chelonia mydas successful Moore et al. 1997 Chelonia mydas Disaggregation Herbst et al. 1998 Chelonia mydas Disaggregation Lu et al. 1999 Chelonia mydas Both, with equal success Work et al. 2009 Chelonia mydas Disaggregation Fukuda et al. 2012 Eretmochelys imbricata Disaggregation Takeshita et al. 2013 Eretmochelys imbricata Disaggregation Wise et al. 2014 Eretmochelys imbricata Disaggregation Fukuda et al. 2014 Lepidochelys olivacea Disaggregation

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Paper Species Medium Results Wolf et al. 1960 Chrysemys picta Medium 199 Callard et al. 1980 Chrysemys picta BME Chow et al. 1985 Chrysemys picta Medium 199 and Coon's modified Ham F12 Clark et al. 1967 Terrapene carolina BME Huang et al. 1967 Terrapene carolina BME Curry et al. 2000 Terrapene carolina DMEM/F12 Shindarov et al. 1962 Testudo graeca Paul's salt solution Fauconnier et al. 1963 Testudo graeca self-made medium and Medium 199 Falcoff et al. 1965 Testudo graeca hydrosylate culture medium Clark et al. 1970 Testudo graeca BME Huang and Clark 1969 Podocnemis unifilis BME Huang and Clark 1969 Podocnemis expansa BME Clark et al. 1970 Podocnemis unifilis BME Milton et al. 2007 Trachemys scripta MEM-L-glutamine with Earl's Salts Nayak et al. 2009 Trachemys scripta MEM-L-glutamine with Earl's Salts Li et al. 2000 Pelodiscus sinensis TC199 medium Li et al. 2010 Pelodiscus sinensis MEM or RPMI 1640 Liu et al. 2012 Pelodiscus sinensis L-15 medium Fu et al. 2013 Pelodiscus sinensis DMEM/F12 or M199 Stephenson et al. 1966 Chelonia mydas Medium 199 Koment et al. 1982 Chelonia mydas MEM Mansell et al. 1989 Chelonia mydas MEM Moore et al. 1997 Chelonia mydas RPMI 1640 Herbst et al. 1998 Chelonia mydas DMEM/F12 Lu et al. 1999 Chelonia mydas RPMI 1640 Lu et al. 2000 Chelonia mydas RPMI 1640 Herbst et al. 2001 Chelonia mydas DMEM/F12 Work et al. 2009 Chelonia mydas Leibowitz with L-glutamine Tan et al. 2010 Chelonia mydas RPMI 1640 Wang et al. 2013 Chelonia mydas RPMI 1640 Fukuda et al. 2012 Eretmochelys imbricata RPMI 1640 Takeshita et al. 2013 Eretmochelys imbricata DMEM Wise et al. 2014 Eretmochelys imbricata RPMI 1640 Fukuda et al. 2014 Lepidochelys olivacea RPMI 1640 Webb et al. 2014 Caretta caretta RPMI 1640

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Paper Species Serum Concentration Paper Species Serum Concentration Wolf et al. 1960 Chrysemys picta 2-5% Milton et al. 2007 Trachemys scripta 10% Callard et al. 1980 Chrysemys picta 10% Nayak et al. 2009 Trachemys scripta 10% Chow et al. 1986 Chrysemys picta 10% Fukuda et al. 2012 Eretmochelys imbricata 10% Chow et al. 1987 Chrysemys picta 10% Takeshita et al. 2013 Eretmochelys imbricata 10% Clark et al. 1967 Terrapene carolina 10% Wise et al. 2014 Eretmochelys imbricata 10% Huang et al. 1967 Terrapene carolina 10% Fukuda et al. 2014 Lepidochelys olivacea 10% Clark et al. 1970 Terrapene carolina 10% Stephenson et al. 1966 Chelonia mydas 20% Curry et al. 2000 Terrapene carolina 5% Koment et al. 1982 Chelonia mydas 5% Shindarov et al. 1962 Testudo graeca 5-10% Mansell et al. 1989 Chelonia mydas 10% Fauconnier et al. 1963 Testudo graeca 5-10% Moore et al. 1997 Chelonia mydas 20% Falcoff et al. 1965 Testudo graeca 5% Herbst et al. 1998 Chelonia mydas 10% Clark et al. 1970 Testudo graeca 10% Lu et al. 1999 Chelonia mydas 10% Li et al. 2000 Pelodiscus sinensis 10% Lu et al. 2000 Chelonia mydas 10% Li et al. 2010 Pelodiscus sinensis 20% Herbst et al. 2001 Chelonia mydas 10% Liu et al. 2012 Pelodiscus sinensis 10% Work et al. 2009 Chelonia mydas 10% Fu et al. 2013 Pelodiscus sinensis 20% Tan et al. 2010 Chelonia mydas 10% Clark et al. 1970 Podocnemis unifilis 10% Wang et al. 2013 Chelonia mydas 10% Huang and Clark 1969 Podocnemis unifilis 10% Webb et al. 2014 Caretta caretta 10% Huang and Clark 1969 Podocnemis expansa 10%

Table 1.4. Summary of published serum concentrations used in the culture of turtle cells.

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Paper Species Incubation Temperature Paper Species Incubation Temperature Callard et al. 1980 Chrysemys picta 30°C Milton et al. 2007 Trachemys scripta 30°C Chow et al. 1985 Chrysemys picta 37°C Nayak et al. 2009 Trachemys scripta 30°C Wolf et al. 1960 Chrysemys picta 19°C Fukuda et al. 2012 Eretmochelys imbricata 26°C Huang et al. 1969 Podocnemis expansa 30°C Takeshita et al. 2013 Eretmochelys imbricata 25°C - 30°C Clark et al. 1970 Podocnemis unifilis 30°C Wise et al. 2014 Eretmochelys imbricata 25°C Huang et al. 1969 Podocnemis unifilis 30°C Fukuda et al. 2014 Lepidochelys olivacea 26°C Clark et al. 1967 Terrapene carolina 23°C Waddell et al. 1965 Chelonia mydas 30°C Curry et al. 2000 Terrapene carolina 28°C Stephenson et al. 1966 Chelonia mydas 37.5°C Huang et al. 1967 Terrapene carolina 23°C Koment et al. 1982 Chelonia mydas 30°C Clark et al. 1970 Terrapene carolina 23°C - 30°C Mansell et al. 1989 Chelonia mydas 30°C Falcoff et al. 1965 Testuda graeca 30°C Moore et al. 1997 Chelonia mydas 30°C Fauconnier et al. 1963 Testuda graeca 25°C - 37°C Herbst et al. 1998 Chelonia mydas 30°C Shindarov et al. 1962 Testuda graeca 37°C Lu et al. 1999 Chelonia mydas 30°C Clark et al. 1970 Testuda graeca 30°C Herbst et al. 2001 Chelonia mydas 30°C Li et al. 2000 Pelodiscus sinensis 25°C Tan et al. 2010 Chelonia mydas 25°C Li et al. 2010 Pelodiscus sinensis 32°C Wang et al. 2013 Chelonia mydas 25°C Liu et al. 2012 Pelodiscus sinensis 30°C Webb et al. 2014 Caretta caretta 30°C Fu et al. 2013 Pelodiscus sinensis 28°C

Table 1.5. Summary of published reported incubation temperatures for turtle cell cultures. If a variety of temperatures was tested, only the optimal temperature(s) was included.

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1.4 References

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Bolten, A. B., L. B. Crowder, M. G. Dodd, S. L. MacPherson, J. A. Musick, B. A. Schroeder, B. E. Witherington, K. J. Long and M. L. Snover (2010). "Quantifying multiple threats to endangered species: an example from loggerhead sea turtles." Frontiers in Ecology and the Environment 9(5): 295-301.

Callard, G. V. (1981). "Aromatization is cyclic AMP-dependent in cultured brain cells." Brain research 204(2): 461-464.

Callard, G. V., Z. Petro and K. J. Ryan (1980). "Aromatization of androgen to estrogen by cultured turtle brain cells." Brain research 202(1): 117-130.

Chow, S., Y. Yen-Chow, H. White and D. Woodbury (1986). "Effects of sodium on iodide transport in primary cultures of turtle thyroid cells." Am. J. Physiol 250: E464-E469.

Chow, S., Y. Yen-Chow, H. White and D. Woodbury (1987). "Effects of 4, 4′-di-isothiocyano-2, 2′-stilbene disulphonate on iodide uptake by primary cultures of turtle thyroid follicular cells." Journal of endocrinology 113(3): 403-412.

Chow, S., Y. Yen-Chow and D. Woodbury (1985). "Water and electrolyte contents, cell pH and membrane potentials of cultured turtle thyroid cells." Journal of endocrinology 104(1): 45-52.

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Clark, H. F., M. M. Cohen and D. T. Karzon (1970). "Characterization of reptilian cell lines established at incubation temperatures of 23 to 36." Experimental Biology and Medicine 133(3): 1039-1047.

Falcoff, E. and B. Fauconnier (1965). "In vitro Production of an Interferon-Like Inhibitor of Viral Multiplication by a Poikilothermic Animal Cell, The Tortoise (Testudo greca)." Experimental Biology and Medicine 118(3): 609-612.

Fauconnier, B. (1963). Annales de l'Institut Pasteur. Eosinophilic nuclear inclusions induced by the multiplication of newcastcle disease virus in tortoise (Testudo graeca) kidney cells.

Fauconnier, B. (1963). Annales de l'Institut Pasteur. Multiplication of mumps virus, vaccinia virus and herpesvirus on tortoise (Testudo graeca) kidney cells cultured by a simplified method.

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Fu, J. P., Z. Luo, Y. Yan, P. F. Zou, S. H. Zhang, Q. W. Qin and P. Nie (2013). "Establishment of a Cell Line from Chinese Soft-shelled Turtle Pelodiscus sinensis with the Practicability of Transfection and Viral Replication." Fish Pathology 魚病研究 48(4): 126-134.

Fukuda, T., M. Katayama, K. Kinoshita, T. Kasugai, H. Okamoto, K. Kobayashi, M. Kurita, M. Soichi, K. Donai and T. Uchida (2014). "Primary fibroblast cultures and karyotype analysis for the olive ridley sea turtle (Lepidochelys olivacea)." In Vitro Cellular & Developmental Biology-Animal 50(5): 381-383.

Fukuda, T., J. Kurita, T. Saito, K. Yuasa, M. Kurita, K. Donai, H. Nitto, M. Soichi, K. Nishimori and T. Uchida (2012). "Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata)." In Vitro Cellular & Developmental Biology-Animal 48(10): 660-665.

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Guo, Q. (2000). "Identification of an interleukin-2 substance in splenocyte culture supernatant of grass carp Ctenopharyngodon idellus and Chinese soft-shelled turtle Trionyx sinensis." Acta hydrobiologica Sinica/Chung-kuo ko hsueh yuan shui sheng sheng wu yen chiu so 25(1): 21-27.

Hahn, G. M. and E. C. Shiu (1983). "Effect of pH and elevated temperatures on the cytotoxicity of some chemotherapeutic agents on Chinese hamster cells in vitro." Cancer Research 43(12 Part 1): 5789-5791.

Harrison, R. G. (1907). "Observations of the living developing nerve fiber." The Anatomical Record 1(5): 116-128.

Hays, G. C. (2008). "Sea turtles: a review of some key recent discoveries and remaining questions." Journal of Experimental Marine Biology and Ecology 356(1): 1-7.

Herbst, L. H., R. Chakrabarti, P. A. Klein and M. Achary (2001). "Differential gene expression associated with tumorigenicity of cultured green turtle fibropapilloma-derived fibroblasts." Cancer genetics and cytogenetics 129(1): 35-39.

Herbst, L. H., J. P. Sundberg, L. D. Shultz, B. A. Gray and P. A. Klein (1998). "Tumorigenicity of green turtle fibropapilloma-derived fibroblast lines in immunodeficient mice." Comparative Medicine 48(2): 162-167.

Hestermann, E. V., J. J. Stegeman and M. E. Hahn (2002). "Serum withdrawal leads to reduced aryl hydrocarbon receptor expression and loss of cytochrome P4501A inducibility in PLHC-1 cells." Biochemical Pharmacology 63(8): 1405-1414.

Huang, C. and H. F. Clark (1967). "Chromosome changes in cell lines of the box turtle (Terrapene carolina) grown at two different temperatures." Canadian Journal of Genetics and Cytology 9(3): 449-461.

Huang, C. and H. F. Clark (1969). "Chromosome studies of the cultured cells of two species of Side-Necked Turtles (Podocnemis unifilis and P. expansa)." Chromosoma 26(3): 245- 253.

Koment, R. W. and H. Haines (1982). "Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle, Chelonia mydas." In Vitro 18(3): 227-232.

Li, X.-l., L.-b. Zeng, Y. Zhang, L. He and Y.-f. Xu (2010). "Report on in vitro Culture of Cell Lines Derived from Different Tissues of Chinese Soft-shelled turtle, Trionyx sinesis." Journal of Hydroecology 2: 021.

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Li, Z. and Q. Zhang (2000). "Establishment and characteristics of heart fibroblast cell line of the soft-shell turtle (Trionyx sinesis)." Chinese journal of veterinary science 21(6): 583-585.

Liu, P.-C., C.-Y. Wang, S.-L. Lin, C.-C. Lin, S.-W. Hung, C.-H. Chang, C.-Y. Tu, M.-H. Chen, Y.-P. Chen and W.-S. Wang (2012). "Establishment of a soft shell turtle, Pelodiscus sinensis, embryo primary cell culture for studies of soft shell turtle poxvirus-like virus replication and characteristics." African Journal of Microbiology Research 6(5): 960-967.

Lu, Y., A. A. Aguirre, T. M. Work, G. H. Balazs, V. R. Nerurkar and R. Yanagihara (2000). "Identification of a small, naked virus in tumor-like aggregates in cell lines derived from a green turtle, Chelonia mydas, with fibropapillomas." Journal of virological methods 86(1): 25-33.

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Rhodin, A., A. D. Walde, B. D. Horne, P. P. van Dijk, T. Blanck and R. Hudson (2011). Turtles in Trouble: The World's 25+ Most Endangered Tortoises and Freshwater Turtles, 2011, IUCN/SSC Tortoise and Freshwater Turtle Specialist Group.

Shindarov, L. (1961). "Tissue culture of kidney epithelium of tortoise (Tenstudo graeca)." Comptes rendus de l'Academie Bulgare des sciences: sciences mathematiques et naturelles 15: 539-542.

Stephenson, N. G. (1966). "Effects of temperature on reptilian and other cells." Journal of Embryology and Experimental Morphology 16(3): 455-467.

Takeshita, S., N. Matsuda, S. Kodama, K. Suzuki and M. Watanabe (2013). "In Vitro Thermal Effects on Embryonic Cells of Endangered Hawksbill Turtle Eretmochelys imbricata." Zoological Science 30(12): 1038-1043.

Tan, F., M. Wang, W. Wang, A. A. Aguirre and Y. Lu (2010). "Validation of an in vitro cytotoxicity test for four heavy metals using cell lines derived from a green sea turtle (Chelonia mydas)." Cell Biology and Toxicology 26(3): 255-263.

Waddell, G. and M. Sigel (1965). Characteristics of kidney cell cultures derived from a marine turtle. Bacterial. Proc.

Wang, H., J. Tong, Y. Bi, C. Wang, L. Guo and Y. Lu (2013). "Evaluation of mercury mediumted in vitro cytotoxicity among cell lines established from green sea turtles." Toxicology in Vitro 27(3): 1025-1030.

Wise, S. S., H. Xie, T. Fukuda, W. D. Thompson and J. P. Wise Sr. (2014). "Hexavalent chromium is cytotoxic and genotoxic to hawksbill sea turtle cells." Toxicology and Applied Pharmacology 279: 113-118.

Wolf, K., M. Quimby, E. Pyle and R. Dexter (1960). "Preparation of monolayer cell cultures from tissues of some lower vertebrates." Science 132(3443): 1890-1891.

Work, T. M., J. Dagenais, G. H. Balazs, J. Schumacher, T. D. Lewis, J.-A. C. Leong, R. N. Casey and J. W. Casey (2009). "In vitro biology of fibropapilloma-associated turtle herpesvirus and host cells in Hawaiian green turtles (Chelonia mydas)." Journal of General Virology 90(8): 1943-1950.

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Chapter 2

Establishment, Characterization, and Toxicological Application of Loggerhead Sea Turtle (Caretta caretta) Primary Skin Fibroblast Cell Cultures

(Submitted to and formatted for Environmental Toxicology and Chemistry)

2.1 Introduction

All seven extant species of sea turtle in the world are listed as vulnerable, endangered, critically endangered, or data deficient (flatback sea turtle, Natator depressus) on the International Union for Conservation of Nature’s (IUCN) Red List of Threatened Species (Pritchard 1996, Wallace, DiMatteo et al. 2011, IUCN 2014). Anthropogenic risks to marine turtles are convoluted; in addition to habitat degradation, boat strikes, and entanglement in fishing gear, exposure to potentially toxic contaminants occurs both in the marine environment and on land (Gibbons, Scott et al. 2000, Hays 2008, Bolten, Crowder et al. 2010). Sea turtles are both migratory and long-lived, increasing chances for long-term exposure to multiple environmental contaminants. Very little is known about the effects of anthropogenic environmental contaminants on all reptile species compared to other taxa (Hopkins 2000). There is a critical need to reliably assess and monitor the effects of environmental pollutants on sea turtle populations within the legal constraints of their threatened or endangered status. Traditional invasive or lethal toxicology testing methods are very seldom suitable to endangered species research (Godard, Smolowitz et al. 2004, Godard, Wise et al. 2006). For this reason, toxicology research in sea turtles so far has focused mainly on contaminant burden analyses in tissues (Alam and Brim 2000, Day, Christopher et al. 2005, Keller, Kannan et al. 2005, Keller, McClellan-Green et al. 2006, Storelli, Barone et al. 2007, Camacho, Boada et al. 2012, Camacho, Luzardo et al. 2013, Faust, Hooper et al. 2014) with rare forays into animal dosing (Lutz and Lutcavage 1989, Lutcavage, Lutz et al. 1995) or egg exposure studies (Fritts and McGehee 1982, Podreka, Georges et al. 1998). Immortal cell lines were developed for virology in the green sea turtle (Chelonia mydas) almost exclusively from animals affected by fibropapillomatosis (Koment and Haines 1982, Mansell, Jacobson et al. 1989, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999) or

Texas Tech University, Sarah J. Webb, December 2014 from embryos (Moore, Work et al. 1997). Pioneering in vitro toxicity testing in sea turtles used these cell lines to examine the effect of organochlorines on cytochrome P450 aromatase (Keller and McClellan-Green 2004) and test metal sensitivity (Tan, Wang et al. 2010, Wang, Tong et al. 2013). Primary skin fibroblasts and embryonic cultures were recently established and partially characterized in the hawksbill sea turtle (Eretmochelys imbricata) and olive ridley sea turtle (Lepidochelys olivacea) (Fukuda, Kurita et al. 2012, Takeshita, Matsuda et al. 2013, Fukuda, Katayama et al. 2014). Primary skin fibroblasts from one Hawksbill sea turtle were subsequently evaluated for chromium toxicity (Wise, Xie et al. 2014) and a preliminary assessment of thermal effects on heat shock protein expression was conducted in embryonic cultures in the same species (Wise, Xie et al. 2014).Additionally,there have been leukocytes cultured from loggerhead turtles (Keller, McClellan-Green et al. 2005, Keller, McClellan-Green et al. 2006), however there are several major differences in the culture of blood cells versus adherent cells reported here. Blood cells typically require mitogen for proliferation and are short-lived. These leukocyte cultures were successfully used to test immune response following exposure to organochlorines (Keller, McClellan-Green et al. 2006).

In vitro toxicology is well established in medicine and pharmacology and relied upon heavily in industry in the case of drug trials. However, mammalian cells are used as the primary model system and very little in vitro research has been conducted using reptilian cells. A series of recent studies (Keller and McClellan-Green 2004, Tan, Wang et al. 2010, Wang, Tong et al. 2013) involved immortal cell lines derived from the same debilitated immature male green sea turtle afflicted with fibropapillomatosis (Lu, Nerurkar et al. 1999). While these studies provide important information and the cell lines are appropriate for virology based research, extrapolation of toxicology data to the species level is limited by the following factors: 1) the diseased state of the source animal, 2) the fact that data derived from a single animal could reflect a phenotypic bias, and 3) the spontaneous (likely disease-induced) immortalization process, which may have caused cellular changes altering the capacity of the cell lines to accurately reflect in vivo processes. This last point is illustrated by the observed lack of cytochrome P450 19 (aromatase) induction, which could infer a missing induction pathway in the immortalized green turtle testis cell line (Keller and McClellan-Green 2004).

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Here, we report on further advances regarding in vitro reptilian toxicology testing, including development and validation of novel methodologies for establishing and characterizing primary sea turtle cell cultures from skin biopsies, coupled with toxicological assessments. These primary cultures are well suited for in vitro research because they originated from skin biopsies of multiple young, healthy captive-reared loggerheads. The use of primary cell cultures circumvents the cellular alterations generally associated with immortalization, and deriving cell cultures from multiple individuals more accurately accounts for intra-species variability.

The first objective of this study was to fully characterize the primary skin fibroblast cell cultures we established from the loggerhead sea turtle (Caretta caretta) by confirming the cell type and determining optimal growth conditions. Characterization is among the first and most critical steps in working with any cell culture (Freshney 2005), yet it is often overlooked in cultures established from wildlife. Four factors known to influence cell growth (medium, serum, temperature, and plate coating/substrate) were tested using a single-factor manipulation or one-variable-at-a-time (OVAT) approach followed by a fractional factorial (FF) design.

The second objective of the study was to adapt standard in vitro toxicological assays to these unique reptilian primary cells. Two cytotoxicity assays, 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH), and a biomarker assay for cytochrome P4501A induction were selected. The MTT assay is based on the conversion of MTT salt to formazan in normal functioning mitochondria(Mosmann 1983). Lactate dehydrogenase is an enzyme normally present in the cytosol and periodically released at low levels through exocytosis. It is released in greater amounts in cells undergoing necrosis, apoptosis, or other mechanisms affecting the cell membrane than in healthy cells(Wolterbeek and Van der Meer 2005). MTT and LDH assays were conducted following exposure to environmentally relevant concentrations of perfluorooctanoic acid (PFOA), a common marine contaminant which has been detected in the serum of sea turtles (O’Connell, Arendt et al. 2010, Keller, Ngai et al. 2012) and is known to cause significant cytotoxicity in mammalian in vitro studies (Freire, Martin et al. 2008). Quantitative real-time PCR (qPCR) was used to quantify the expression of cytochrome P4501A5 (CYP1A5) following exposure to benzo(a)pyrene 21

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(B(a)P), a prototypical polycyclic aromatic hydrocarbon (PAH) and common marine contaminant (Miller and Ramos 2001). CYP1A5, a newly cloned reptilian gene belonging to the CYP1A subfamily (Wiggins 2011), is known to play a critical role in the metabolism, detoxication, and bioactivation of PAHs in mammals (Conney 1982). Polycyclic aromatic hydrocarbons have been detected in plasma or tissues of green, loggerhead, Kemp’s ridley (Lepidochelys kempii), and leatherback (Dermochelys coriacea) sea turtles, and in loggerhead eggs (Hall, Belisle et al. 1983, Godley, Gaywood et al. 1998, Alam and Brim 2000, Camacho, Boada et al. 2012, Camacho, Luzardo et al. 2013).

2.2 Experimental Methods Sampling Methods and Permits

Cell cultures were derived from skin biopsies collected from healthy captive-reared animals held at the NOAA/NMFS Sea Turtle Facility in Galveston, Texas. All turtles sampled were between two and four years old. Biopsies were collected under the supervision of a veterinarian as 6-8 mm punches from the hind flipper and stored in cold, supplemented shipping medium immediumtely upon collection (RPMI 1640 medium (Mediumtech) 1.0% penicillin-streptomycin (Mediumtech), 1.0% Amphotericin B (Mediumtech)). Samples were shipped cold overnight to The Institute of Environmental and Human Health (TIEHH), Texas Tech University, Lubbock, Texas.

All biopsies were collected under U.S. Fish and Wildlife Service Endangered Species Act Section 10a(1)a Scientific Research Permit TE-676379-4 and TE-676379-5, and Florida Fish and Wildlife Conservation Commission Marine Turtle Permit #MTP- 015. All research complied with all institutional animal care guidelines.

Establishment of Primary Skin Fibroblast Cultures

Upon arrival of biopsies at Texas Tech University, shipping medium was exchanged for 10 mL phosphate buffered saline (PBS) (HyClone) supplemented with 1.0% penicillin streptomycin and 1.0% Amphotericin B (Mediumtech). After a 30-min

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4°C refrigeration, biopsies were briefly dipped in 70% ethanol three times for sterilization. The tissue was minced by scalpel in 30°C complete cell culture medium (RPMI1640 medium, 10% bovine calf serum, 1.0% penicillin streptomycin, 1.0% Amphotericin B) then transferred to a vented 25 cm2 cell culture flask (Corning). Explants were arranged evenly on the growth surface and flasks were maintained inverted without medium for 23 hours in a Steri-Cult CO2 incubator (Thermo Scientific) at 30°C,

86% relative (RH), and 5% CO2. Incubation then continued after addition of complete cell culture medium (shipping medium plus ~ 10% bovine calf serum (BCS)) and reversion of flasks. Cells were harvested at or near cell confluence, counted, and transferred either to a 75 cm2 or 150 cm2 vented flask (Corning).

Three alternative methods of explantation were also tested in addition to the above standard laboratory explant procedure. These methods included (1) a scratched flask surface, (2) serum coated flasks, and (3) varying timing of first medium change (See supporting information).

Optimization of Primary Culture Growth Conditions

Growth curve assays were used to test four critical factors contributing to cell growth: 1) medium type (RPMI 1640, MEM/EBSS (HyClone), or DMEM/F12 (HyClone)), 2) growth surface coating (tissue culture (TC)-treated, TC-treated with gelatin (0.1%), TC-treated with collagen IV (2.5 μg/cm2, Corning), or TC-treated with fibronectin (2.5 µg/cm2, Corning)), 3) medium concentration of BCS (~5%, ~10%, or ~15%), and 4) incubation temperature (25°C, 30°C, or 35°C ), as seen in Table 2.1. For each of the 10 factorial combinations tested (Table 2.1), growth curves were performed ≥ 3 times using cells derived from different animals to incorporate potential biological variability. Initially, tests were performed using a single-factor manipulation or one- variable-at-a-time (OVAT) approach, in which only one variable was manipulated from the standard laboratory cell growth conditions for each test. For growth curve assays and for each factor tested, wells were seeded in triplicate with 5 X 104 cells into seven 12- well plates (Greiner Bio One), in triplicate for each factor. Medium was replaced every two days. Cells were counted from one plate every five days until the end of the

Texas Tech University, Sarah J. Webb, December 2014 experiment at 35 days. Optimal conditions were determined based on statistical differences in final cell counts at each time point (p≤0.05). The ten OVAT combinations were analyzed in JMP ® 8.0 statistical software by one-way analysis of variance (ANOVA). Significant differences and overall averages were used to select the two best conditions for each factor.

A fractional factorial (FF) design was then applied by selecting the best performing options for each factor examined during the OVAT testing approach. The FF design incorporated two levels each for medium, temperature, and coating/substrate but only one level for temperature since OVAT testing revealed that 30°C greatly outperformed the other two temperatures. The design matrix was created by JMP ® 8.0 3-1 following a 2III format; i.e., of the eight combinations possible for three factors at two levels each, the design captured four of these, described as the “first fraction,” with an acceptable compromise between confounding effects and number of combinations tested, such that the main effects could be detected (Ryan 2007). Because three of the four combinations generated by the FF design had already been tested during OVAT testing, the only remaining combination in the matrix that required testing was that of MEM/EBSS with ~15% serum, with a 0.1% gelatin-coating on TC-treated surface, and 30°C incubation.

Immunocytochemistry

Cell identity was confirmed by immunocytochemistry (ICC) using an antibody against vimentin, a type III intermediumte filament, known to be expressed in fibroblasts (Moll, Franke et al. 1982). At 90-95% confluence, cells were rinsed with room- temperature PBS and fixed in 100% ice-cold methanol (Fisher) for 20 minutes at -20C. Methanol was replaced by PBS and plates were kept at 4-5°C until ICC analysis. For ICC, PBS was replaced with PBS containing 0.1% Tween 20 (PBST) (Genetex). Plates were rocked at room temperature for 10 minutes. PBS was aspirated, and a rabbit polyclonal antibody against vimentin (Abcam ab71144) was added, at dilutions of 1:50 and 1:100, to three wells for each concentration. Cells were tested separately for the presence of cytokeratin 8 and 18, a type of intermediumte filament characteristic of

Texas Tech University, Sarah J. Webb, December 2014 epithelial cells (Moll, Franke et al. 1982), with the primary antibody NCL-L-5D3 (Novocastra) at a 1:100 dilution. Samples were screened for autofluorescence using a PBST-only treatment (no primary antibody). Plates were covered and rocked at 4C overnight, then rinsed with PBS three times and incubated with a rhodamine-conjugated, goat-anti-rabbit IgG Red-X (Molecular Probes #R6394) secondary antibody at a 1:250 dilution for 1 hour. The nuclear counterstain 4',6-diamidino-2-phenylindole (DAPI) (VWR) was added to all wells after a PBST rinse for orientation purposes.

Karyotyping

Karyotyping followed the methods of Herbst et al.(Herbst, Sundberg et al. 1998). Cells were cultured from a two year old animal (sex undetermined) until they exhibited abundant mitotic activity, at which point demecolcine (Fisher) was added to arrest cells in metaphase. Cells were harvested and fixed using 3:1 methanol/glacial acetic acid. Following fixation, cells were stained with Giemsa and analyzed with a Zeiss Axioplan2 microscope equipped with Ikaros image analysis software v5.2 (MetaSystems GmbH).

MTT assays

MTT assays were optimized for use with our characterized cell cultures. For each of six animals, cells were seeded with 5 X 104 cells/well in TC-treated 96-well plates (Greiner Bio One) and maintained for 48 hours at 30C to allow for cell attachment. Cells were then dosed in triplicate with PFOA (Sigma Aldrich) at concentrations of 0.05 μM, 0.5 μM, 5.0 μM, 50 μM, and 500 μM for either 72 or 96 hours. This dose range encompasses concentrations found in sea water, prey items and sea turtle tissues(Yamashita, Kannan et al. 2005, Gulkowska, Jiang et al. 2006, O’Connell, Arendt et al. 2010, Keller, Ngai et al. 2012). The stock solution of PFOA, which was serially diluted and used in dosing, was analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and was confirmed to be within 3.0% of the desired concentration (additional method details available in supporting information)). Two controls were used, one with vehicle only in medium (0.087% dimethyl sulfoxide,

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DMSO) and one of medium only. After treatment period, medium containing PFOA was removed, 100 µL medium was added to each well, followed by 10 µL of MTT (CalBiochem, prepared as 5 mg/mL in PBS, sterile filtered). Incubation time for MTT metabolism was optimized for C. caretta cell cultures by testing 4, 12, and 24 hour time points. Twenty-four hours was found to be optimal to measure absorbance. Following 24 hour MTT exposure, medium was removed from the plate and replaced with 100 µL DMSO to solubilize the formazan product. The plate was covered to eliminate light exposure, rocked for 30 min, and analyzed for absorbance at 570 nm and at a reference wavelength of 650 nm using a BioTek Synergy™ 4 Microplate Reader and BioTek Gen5™ software. Viability relative to the control was calculated and one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc analysis was performed using R (R Foundation for Statistical Computing, Vienna, Austria) to compare average viability for each dose.

LDH Assays

A Lactate Dehydrogenase Cytotoxicity Assay Kit (Cayman Chemical 10008882) was utilized to quantify LDH concentration in cells from five individuals. Because this assay requires only medium, it was used in conjunction with MTT assays on cells dosed with PFOA as previously described. Medium was collected prior to the addition of MTT to wells. The tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H- tetrazolium chloride (INT) is metabolized to formazan in the presence of LDH, and cytotoxicity information is thus obtained by spectrophotometry. Absorbance was read on a BioTek Synergy™ 4 Microplate Reader at 490 nm. Standard curves were plotted using absorbance as a function of LDH concentrations and used to calculate LDH activity in each sample (μU/ml). One way ANOVA was used to determine whether statistical differences in LDH activity occurred using the ISwR package in R (p≤0.05) (R Foundation for Statistical Computing, Vienna, Austria), and a Tukey’s test was used for post-hoc analysis as necessary.

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Quantitative RT-PCR

For each of six animals, ~12 x 106 fibroblasts were divided equally between five

T-75 flasks and incubated in our optimized culture conditions (30°C, 5.0% CO2) for 48 hours to allow cell attachment. One control flask was exposed to 0.087% DMSO (carrier) only and four treatment flasks were exposed to B(a)P at 0.01 μM, 0.1 μM, 1.0 μM, or 10 μM for 72 hours, which represents what has been measured in sea turtle tissues(Hall, Belisle et al. 1983, Camacho, Boada et al. 2012, Camacho, Luzardo et al. 2013). The stock solution of B(a)P, which was used to make serial dilutions for dosing, was analyzed for accuracy using liquid chromatography with UV detection (LC-UV). Accuracy was confirmed within 3.5% of the target concentration (additional method details available in supporting information). Total RNA was isolated using a NucleoSpin®RNAII kit (Clontech, Foster City, CA) and integrity was evaluated by electrophoresis on 1% agarose gels in 1X MOPS buffer.

Quantitative reverse transcription PCR (qPCR) primers were designed for the loggerhead CYP1A5, actin, and 18S genes (Genbank accession number HQ293214, HQ828316.1, and HQ914786.1, respectively; Wiggins 2011). Fluorescence-based qRT- PCR was performed with the Rotor-Gene™ SYBR Green RT-PCR kit (Qiagen) using the liquid handling robot CAS-1200™ and the Rotor-Gene 6000™ (Corbett Research, Mortlake, New South Wales, Australia). Each sample was run in triplicate.

LinRegPCR software was used to generate a cycle threshold (Ct) and amplification efficiencies for each sample(Ramakers, Ruijter et al. 2003). 18S was selected as the best normalizing gene when compared to actin based upon the least amount of change in Ct values between tissue types or treatment groups and due to similar amplification efficiencies to CYP1A5 (between 1.7-1.8)(McCurdy, McGrath et al. 2008). qRT-PCR analysis of gene expression was carried out using the relative -ΔΔCt quantification delta-delta Ct method (2 )(Livak and Schmittgen 2001). For all the treated groups, the expression ratio results were compared to control and tested for significance by a randomization test using the relative expression software tool

REST©(Pfaffl, Horgan et al. 2002). A one-way ANOVA was also used to analyze ∆∆Ct

Texas Tech University, Sarah J. Webb, December 2014 values for significance (p≤0.05) between treatment groups. All statistical analysis was done using R (R Foundation for Statistical Computing, Vienna, Austria).

2.3 Results

Optimization of Growth Conditions

Our standard explantation method was the simplest and most cost- and time- effective of all four methods we tested and was selected for use thereafter. For each of these methods, time until confluence was recorded (Table S1). Cell increases were observed over the course of 35 days for all medium tested (Figure 2.1 A). RPMI and MEM/EBSS yielded slightly higher averages than DMEM/F12 and were selected for the FF design. Serum concentrations of ~10% and ~15% were retained for the FF design based on observed higher yields (Figure 2.1 B). Due to cell count results at or near 0 by Day 35 for 25 and 35C, 30°C was the single option retained for the FF design (Figure 2.1 C). TC-treated polystyrene growth surface yielded the largest cell counts (Figure 2.1 D) and was the most cost- and time-effective of all options tested. Statistically higher cell counts were obtained at days 25, 30 and 35 compared to the gelatin, fibronectin, and collagen treatments. Among options for additional coating only gelatin produced an increase in cell numbers over the course of 35 days. Thus, the TC-treated surface without additional coating and the TC-treated surface with 0.1% gelatin coating were retained for the FF design (Table 2.2). After statistical analyses, graphical representation of data, and cost considerations, the optimal combination of conditions was selected as RPMI 1640, 30°C incubation, ~10% serum, and a TC-treated surface.

Confirmation of Cell Type by Morphology and Immunocytochemistry

Morphological characteristics of fibroblasts were consistent with what has been previously described in green sea turtle fibroblasts(Lu, Nerurkar et al. 1999, Work, Dagenais et al. 2009), e.g. a spindle shape and varied branching (Figure 2.2 A). Cells typically self-organized into densely packed, parallel arrangements upon reaching confluence (Figure 2.2 B). ICC confirmed the presence of vimentin in the cells’

Texas Tech University, Sarah J. Webb, December 2014 cytoplasm (Figure 2 C), providing further confirmation of fibroblast cell type. The anti- cytokeratin antibody used as the negative control, which is specific for epithelial cells, demonstrated no fluorescence after incubation. This experiment served to confirm the fibroblast cell traits as well as demonstrate the purity of cell culture.

Karyotyping

Karyotyping showed that the diploid number of the loggerhead sea turtle is 2n=56, including 24 macrochromosomes (12 pair) and 32 microchromosomes (16 pair) and no sex chromosomes, which is standard for turtle species which exhibit temperature dependent sex determination(Standora and Spotila 1985) (Figure 2.3 A,B). Chromosomes were counted in 24 cells from one individual, with 19 cells exhibiting 2n=56 and 6 incomplete cells with either 55 or 54 chromosomes.

Cytotoxicity Assays

Significant cytotoxicity occurred in cells exposed to PFOA at the highest dose (500 μM) for both MTT assays (n=6 animals, 72 h p≤0.001, 96 h p≤0.001) and LDH assays (n=5 animals, 72 h p≤0.05, 96 h p≤0.01) (Figure 2.4 A,B).

Biomarker Expression (qPCR)

CYP1A5 induction was detected in primary skin fibroblasts after exposure to 0.1 µM, 1 µM, and 10 µM B(a)P (p≤0.001) for 72 hours (Figure 2.5). No differences were observed between treatments.

2.4 Discussion

Our optimal culture conditions were similar to the few previously described chelonian cell culture methods, with the biggest difference being the range of temperatures at which optimal proliferation occurred in other species. Previous literature suggests that temperatures at or slightly above the source animals’ preference are suitable for reptilian cell culture work, and that an optimal temperature would occur between approximately 23°C and 37°C for sea turtles (Stephenson 1966, Wolf 1979, Wallace and

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Jones 2008). Our findings indicated that 30°C is the preferred growth temperature for primary loggerhead fibroblast sea turtle skin cultures and corresponds to the 28-30°C rearing temperature at the NOAA Sea Turtle Facility. Immortal green sea turtle cells are reported to grow well at 25-30°, primary hawksbill and olive ridley sea turtle cells at 26°C, and loggerhead sea turtle leukocytes at 30°C though proliferation was reported at a wide range of other temperatures (Koment and Haines 1982, Mansell, Jacobson et al. 1989, Moore, Work et al. 1997, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999, Keller, McClellan-Green et al. 2005, Keller, McClellan-Green et al. 2006, Fukuda, Kurita et al. 2012, Takeshita, Matsuda et al. 2013).

Reptilian cell cultures reported in the literature utilized cell medium designed for mammalian cells. Multiple medium options have been examined for sea turtle cell cultures, including Medium 199, Minimum Essential Medium (MEM), Dulbecco’s Modified Eagle Medium (DMEM), Leibovitz L-15, F-10 nutrient mix, F-12 nutrient mix, Basal Medium Eagle (BME), and RPMI 1640 (Koment and Haines 1982, Mansell, Jacobson et al. 1989, Moore, Work et al. 1997, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999, Fukuda, Kurita et al. 2012). Our results show that more than one medium could be appropriate for loggerhead primary skin fibroblasts. This may be attributed to similarities in composition between RPMI 1640, MEM/EBSS, and DMEM/F12, although DMEM/F12 and RPMI 1640 have additional protein components that are absent in MEM (Freshney 2005).

A concentration of 10% calf serum was optimal for all previous sea turtle cell cultures (Mansell, Jacobson et al. 1989, Moore, Work et al. 1997, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999, Fukuda, Kurita et al. 2012). Thus our optimal BCS concentrations of ~10% and ~15% were comparable and in agreement with existing literature. Results strongly suggested that loggerhead primary fibroblasts are serum- dependent, as medium without serum (data not shown) resulted in complete cell death.

Proper attachment of cells to the growth surface is essential for normal cell physiology (Ruoslahti and Pierschbacher 1987). The polystyrene surface of cell culture dishes and flasks is a poor substrate for cell adhesion. Charged functional groups are added on the polystyrene to increase cell adhesion in TC treated cell culture dishes and

Texas Tech University, Sarah J. Webb, December 2014 flasks (Curtis, Forrester et al. 1983). Tissue adhesion to the flask surface seems to be a critical factor in initiating cell proliferation, and we investigated several modifications to the surface of tissue culture treated cell culture flasks known to increase the likelihood of tissue attachment for fibroblasts in other species (Grinnell, Feld et al. 1980). We observed that the TC treated flask surfaces outperformed all other treatments tested.

Time until confluent monolayers are produced is markedly longer in our loggerhead cultures, at 45 days from establishment of tissue explants until first passage, compared to 7-12 days for immortal green sea turtle cultures, and 14 days for primary hawksbill cultures(Koment and Haines 1982, Moore, Work et al. 1997, Lu, Nerurkar et al. 1999, Fukuda, Kurita et al. 2012). The shorter time for green sea turtle is likely due to increased growth rates in immortal cells compared to primary cells. The difference in cell culture vessels between our study (15 cm2 flasks) and the hawksbill study (35 mm, i.e. 9.62 cm2 dishes) likely accounts for differences in time until confluence, along with species specificity. Reptilian cultures have slower growth rates than mammalian cells which tend to reach confluence in 3-7 days, as noted by both Stephenson (Stephenson 1966), in cells from the painted turtle, and Moore (Moore, Work et al. 1997) from the green sea turtle. This emphasizes the differences in physiology between mammalian and reptilian cells, highlighting the need for reptilian cell lines in particular, to serve as a platform for toxicological studies.

Our karyotype results showed similar chromosome number to karyotypes reported for the green and hawksbill sea turtles. The first reported sea turtle karyotype for the green sea turtle described heterogametic chromosomes, with females 2n=55 and males 2n=56 (Makino 1952). Subsequent studies disproved the heterogameticity of the species, but confirmed a diploid number of 55 or 56 for the species (Bickham, Bjorndal et al. 1980, Koment and Haines 1982, Moore, Work et al. 1997, Herbst, Sundberg et al. 1998). Fukuda and colleagues (Fukuda, Kurita et al. 2012, Fukuda, Katayama et al. 2014) reported 2n=56 in the hawksbill and olive ridley sea turtles, identical to our findings in the loggerhead (unknown sex).

Our data show that LDH and MTT cytotoxicity assays along with CYP1A gene expression quantitation are viable methods for investigating the impact of contaminant

Texas Tech University, Sarah J. Webb, December 2014 exposure to sea turtle cells. MTT assays have been used successfully in toxicity testing in immortal green sea turtle cell lines and in loggerhead leukocytes, although with a markedly shorter incubation time of 4 hours (Keller, McClellan-Green et al. 2005, Tan, Wang et al. 2010, Wang, Tong et al. 2013). In our experience, MTT assay optimization for primary loggerhead fibroblasts required a longer incubation time of 24 hours to allow the cells to fully metabolize MTT and delineate between control and dosed cells, highlighting a critical need for species and cell specific optimizations of in vitro toxicity assays. Standard LDH methodologies worked well for our cultures. Gene expression of CYP1A5 via qPCR analysis used species-specific loggerhead sea turtle CYP1A5, 18S, β- actin primers previously cloned in our laboratory (Wiggins 2011). The lowest adverse effect level (LOAEL) for PFOA detected here in MTT and LDH assays, 500 μM (2.07 mg/g), is higher than PFOA levels reported in sea turtle tissues (up to 3.78 ng/g in some species) (O’Connell, Arendt et al. 2010, Keller, Ngai et al. 2012). This does not necessarily indicate that PFOA levels found in tissues are not harmful, merely that the levels are not high enough to directly cause cell death in fibroblasts in vitro within our tested exposure time (72 hour). Research measuring PFCs in sea turtle tissues has indicated that levels are well above those known to cause adverse health effects in other species, including immune and neurobehavioral effects (Keller, Ngai et al. 2012), and further research is warranted to elucidate the exact impact of these contaminants to these animals. We observed significant CYP1A5 induction in skin fibroblasts following a 72 hour exposure to B(a)P starting at 0.1 μM. Reports on the in vitro expression and induciblity of CYP1A in fibroblasts from terrestrial mammals are varied (Kim, Deboni et al. 1997, Gradin, Toftgård et al. 1999, Akintobi, Villano et al. 2007, Henry, Welle et al. 2009). Interestingly, CYP1A induction in skin fibroblasts has been reported in marine mammals (Godard, Smolowitz et al. 2004, Fossi, Casini et al. 2008). The establishment and characterization of primary skin cultures in the loggerhead open a much needed avenue to further investigate the expression of CYP1A in these taxa. Given the protected status of the loggerhead and other sea turtles, in vitro research is currently among the few viable options for studying these organisms with a high degree of experimental control. Tissue culture techniques provide much-needed tools for

Texas Tech University, Sarah J. Webb, December 2014 the investigation of toxicological and pathological threats in these and other protected species (Godard-Codding, Clark et al. 2011), and the characterization of cell cultures is a critical first step in that process. To date, comparatively little research has been done toward the development of cell culture models for reptiles, leaving the state of knowledge far behind what is known for mammalian and even some invertebrate cells. The results from this research suggest that by minimally invasive sampling and the use of the optimal conditions described, researchers now have the ability to use these primary cell cultures for a variety of purposes, including cytotoxicity testing. In vitro methods also have the advantage of reproducibility within and between laboratories, and we expect that our characterization and preliminary toxicological work will enable diverse research on a variety of threats to the loggerhead. Growth conditions such as percent serum in the medium, temperature, confluence, can have a major impact on the outcome of cytotoxicity assays (Raz, Fogler et al. 1979, Hahn and Shiu 1983, Hestermann, Stegeman et al. 2002). Standard and consistent growth conditions are critical for replicable, reliable cytotoxicity data. This necessitates the establishment of standardized culture conditions to allow for accurate and repeatable assays among researchers. To the best of our knowledge, our research represents the most thorough characterization and optimization of any sea turtle primary adherent cell cultures. The establishment of these primary cell cultures from healthy animals provides new avenues of research in reptilian cell biology and toxicology. For example, the cultures we established can be used to obtain toxicity information on contaminants of current environmental concerns, such as crude oil exposure in marine turtles. Furthermore, our methodology is applicable to other protected species including the critically endangered Kemp’s ridley sea turtle (L. kempii) from which skin biopsies can be collected.

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Figure 2.2. Caretta caretta primary skin fibroblasts. A) Fibroblasts proliferating from an explant (100X, scale bar = 20μm); B) Fibroblasts in cell culture (100X, scale bar = 10μm); C) Vimentin labeling by immunocytochemistry in Caretta caretta primary skin fibroblasts (100X, scale bar = 50μm). Vimentin is visualized using a rhodamine-conjugated, goat-anti-rabbit IgG Red-X (red), and DAPI counterstained nuclei (blue). Images were captured by deconvolution fluorescence microscopy using a Hamamatsu Orca – ER high-speed camera on an Olympus IX70 inverted microscope (Olympus, Tokyo, Japan) at 100X, and analyzed using Simple PCI software.

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2.5 Supporting Information

Alternative Explant Methods We tested four methods for explant establishment, to our knowledge the first time such optimization has been conducted for reptilian cells. We found no significant differences among establishment methods and therefore retained our original methodology as the simplest and most cost- and time-effective method. In addition to our standard explant methodology, referred to as Method A, three other methods were tested as follows: Method B was identical to Method A except for the following modifications. The flasks’ growth surface was scratched with a Pasteur pipette in a broad wave pattern and then prepped with 2 mL complete cell culture medium for approximately 30-60 min(Ezaz, O’Meally et al. 2008). Medium was removed prior to placement of explants along the scratch marks. Flasks were then left under a sterile hood with loose caps for 30 min before the addition of 5 mL medium and transfer to cell culture incubator. Method C was identical to Method A except for the following modifications recommended by Dr. Erica Bruce (Baylor University, personal communication). Cell culture flask surfaces were coated with 3-5 mL BCS and placed in a 30° incubator for 1- 1.5 hours. Following incubation remaining BCS was aspirated and flasks were left in a sterile hood overnight with lids open under UV light prior to the addition of tissue the following morning. Method D was identical to Method A except for the following modifications recommended by Dr. Thierry Work (United States Geological Services, personal communication). Biopsy tissue was minced into smaller pieces, approximately 0.5-1 mm. Additionally, instead of removing medium and inverting tissue culture flasks, 3 mL of medium was left in flasks and flasks were left right side up. Flasks were then left undisturbed in the incubator for two days. No statistically significant differences in cell yields or time to first passage of cells were observed between the four methods of establishment tested (Table S1). Method A is the simplest and most cost- and time-effective method and was selected for use thereafter.

Cell Counts and Population Doubling Levels

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All cell counts were performed with a Coulter Z2 Particle Count and Size Analyzer set with a size range of 10 µm to 27.70 µm as suitable for fibroblast counts. The population doubling time (PDT) was calculated for these cultures to be 8.5 days (204 hours). This was incorporated into the first calculation of population doubling level (PDLi) following harvesting of cells from explants in the formula PDLi = ; where T= time passed in days since cells were 50% confluent until the first passage at 90% confluency, and 1 is a constant. The constant 1 accounts for the migration of cells from explants to establishment on the flask surface. This formula was designed specifically for these cells as there were no appropriate formulas available to calculate PDLi for primary reptilian cell cultures. Subsequent PDLs were calculated following each passage using the formula

PDL = 3.32 (log N – log N0) + PDLi; where N = final cell count, N0 = original cell count, and PDLi = original PDL(Hayflick 1973). All assays were performed using only cell cultures with a PD of 15 or lower.

Analytical Analysis of Dosing Solutions B(a)P and PFOA stock concentrations were verified by chromatography. Dosing stocks in DMSO were diluted in LC-MS grade methanol to a concentration appropriate for instrumental analysis. B(a)P was quantified using HPLC-UV. Chromatography was conducted with a C18 column (Pinnacle D8, 5µm, 250x3.2mm; Restek, Bellefonte, PA) and isocratic elution with 80:20 acetonitrile:water, and the UV detector was set at 254 nm. PFOA was quantified with a triple quadrupole LC-MS/MS. Chromatography used a gradient elution of water (plus 20mM ammonium acetate):methanol and a Gemini-NX C18 column (3µm, 150x2.0mm; Phenomenex, Torrance, CA), and mass spectrometry was performed using negative electrospray ionization. Calibration curves used for quantitation contained at least five standards and had r2 values of ≥0.995. Concentrations were determined to be 103.5% and 102.7% of nominal concentrations for B(a)P and PFOA, respectively.

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Average Days Before Average Cell Count Standard Error of the First Passage at First Passage Mean Method A (n=10) 45 75,727 13,892 Method B (n=10) 60 74,264 11,733 Method C (n=10) 52 68,092 7,334 Method D (n=10) 44 72,713 10,091

Table S1. Comparison of explant methods, including time to first passage and number of cells yielded at first passage.

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2.6 References

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Day, R. D., S. J. Christopher, P. R. Becker and D. W. Whitaker (2005). "Monitoring mercury in the loggerhead sea turtle, Caretta caretta." Environmental Science & Technology 39(2): 437-446.

Ezaz, T., D. O’Meally, A. E. Quinn, S. D. Sarre, A. Georges and J. A. M. Graves (2008). "A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)." Cytotechnology 58(3): 135-139.

Faust, D. R., M. J. Hooper, G. P. Cobb, M. Barnes, D. Shaver, S. Ertolacci and P. N. Smith (2014). "Inorganic elements in green sea turtles (Chelonia mydas): Relationships among external and internal tissues." Environmental Toxicology and Chemistry.

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Freire, P. F., J. M. P. Martin, O. Herrero, A. Peropadre, E. de la Pena and M. J. Hazen (2008). "In vitro assessment of the cytotoxic and mutagenic potential of perfluorooctanoic acid." Toxicology in Vitro 22(5): 1228-1233.

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cultures from the hawksbill sea turtle (Eretmochelys imbricata)." In Vitro Cellular & Developmental Biology-Animal 48(10): 660-665.

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Godard, C., S. Wise, R. Kelly, B. Goodale, S. Kraus, T. Romano, T. O’Hara and J. Wise Sr (2006). "Benzo [a] pyrene cytotoxicity in right whale (Eubalaena glacialis) skin, testis and lung cell lines." Marine Environmental Research 62: S20-S24.

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Godley, B., M. Gaywood, R. Law, C. McCarthy, C. McKenzie, I. Patterson, R. Penrose, R. Reid and H. Ross (1998). "Patterns of marine turtle mortality in British waters (1992–1996) with reference to tissue contaminant levels." Journal of the Marine Biological Association of the United Kingdom 78(03): 973-984.

Gradin, K., R. Toftgård, L. Poellinger and A. Berghard (1999). "Repression of dioxin signal transduction in fibroblasts Identification of a putative repressor associated with Arnt." Journal of Biological Chemistry 274(19): 13511-13518.

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Gulkowska, A., Q. Jiang, M. K. So, S. Taniyasu, P. K. Lam and N. Yamashita (2006). "Persistent perfluorinated acids in seafood collected from two cities of China." Environmental Science & Technology 40(12): 3736-3741.

Hall, R. J., A. A. Belisle and L. Sileo (1983). "Residues of petroleum hydrocarbons in tissues of sea turtles exposed to the Ixtoc I oil spill." Journal of Wildlife Diseases 19(2): 106-109.

Hayflick, L. (1973). Subculturing Human Diploid Fibroblast Cultures. Tissue Culture, Methods and Applications. J. Paul F. Kruse and J. M.K. Patterson. New York, New York, Academic Press: 220.

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Henry, E. C., S. L. Welle and T. A. Gasiewicz (2009). "TCDD and a putative endogenous AhR ligand, ITE, elicit the same immediumte changes in gene expression in mouse lung fibroblasts." Toxicological sciences: kfp285.

Hopkins, W. A. (2000). "Reptile toxicology: Challenges and Opportunities on the Last Frontier in Vertebrate Ecotoxicology." Environmental Toxicology and Chemistry 19(10): 2391-2393.

IUCN. (2014). "International Union for the Conservation of Nature Red List." Retrieved January 14, 2014, 2014, from http://www.iucnredlist.org/

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Keller, J. M., K. Kannan, S. Taniyasu, N. Yamashita, R. D. Day, M. D. Arendt, A. L. Segars and J. R. Kucklick (2005). "Perfluorinated compounds in the plasma of loggerhead and Kemp's ridley sea turtles from the southeastern coast of the United States." Environmental Science & Technology 39(23): 9101-9108.

Keller, J. M. and P. McClellan-Green (2004). "Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line." Marine Environmental Research 58(2): 347-351.

Keller, J. M., P. D. McClellan-Green, J. R. Kucklick, D. E. Keil and M. M. Peden-Adams (2006). "Effects of organochlorine contaminants on loggerhead sea turtle immunity: comparison of a correlative field study and in vitro exposure experiments." Environmental Health Perspectives: 70-76.

Keller, J. M., P. D. McClellan-Green, A. M. Lee, M. D. Arendt, P. P. Maier, A. L. Segars, J. D. Whitaker, D. E. Keil and M. M. Peden-Adams (2005). "Mitogen- induced lymphocyte proliferation in loggerhead sea turtles: comparison of methods and effects of gender, plasma testosterone concentration, and body condition on immunity." Veterinary immunology and immunopathology 103(3): 269-281.

Keller, J. M., L. Ngai, J. B. McNeill, L. D. Wood, K. R. Stewart, S. G. O'Connell and J. R. Kucklick (2012). "Perfluoroalkyl contaminants in plasma of five sea turtle species: Comparisons in concentration and potential health risks." Environmental Toxicology and Chemistry 31(6): 1223-1230.

Kim, P. M., U. Deboni and P. G. Wells (1997). "Peroxidase-dependent bioactivation and oxidation of DNA and protein in benzo [a] pyrene-initiated micronucleus formation." Free Radical Biology and Medicine 23(4): 579-596.

Koment, R. W. and H. Haines (1982). "Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle, Chelonia mydas." In Vitro 18(3): 227-232.

Livak, K. J. and T. D. Schmittgen (2001). "Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method." Methods 25(4): 402- 408.

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Lutcavage, M., P. Lutz, G. Bossart and D. Hudson (1995). "Physiologic and clinicopathologic effects of crude oil on loggerhead sea turtles." Archives of Environmental Contamination and Toxicology 28(4): 417-422.

Lutz, P. L. and M. Lutcavage (1989). The effects of petroleum on sea turtles: Applicability to Kemp's ridley. Galveston, Texas A&M University Sea Grant Program.

Makino, S. (1952). "The chromosomes of the sea turtle, Chelonia japonica, with evidence of female heterogamety." Annotationes Zoologicae Japonenses 25(1): 250-257.

McCurdy, R. D., J. J. McGrath and A. Mackay-Sim (2008). "Validation of the comparative quantification method of real-time PCR analysis and a cautionary tale of housekeeping gene selection." Gene Therapy & Molecular Biology 12: 15- 24.

Miller, K. P. and K. S. Ramos (2001). "Impact of cellular metabolism on the biological effects of benzo [a] pyrene and related hydrocarbons." Drug Metabolism Reviews 33(1): 1-35.

Moll, R., W. W. Franke, D. L. Schiller, B. Geiger and R. Krepler (1982). "The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells." Cell 31(1): 11-24.

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Mosmann, T. (1983). "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays." Journal of Immunological Methods 65(1): 55-63.

O’Connell, S. G., M. Arendt, A. Segars, T. Kimmel, J. Braun-McNeill, L. Avens, B. Schroeder, L. Ngai, J. R. Kucklick and J. M. Keller (2010). "Temporal and spatial trends of perfluorinated compounds in juvenile loggerhead sea turtles (Caretta caretta) along the east coast of the United States." Environmental Science & Technology 44(13): 5202-5209.

Pfaffl, M. W., G. W. Horgan and L. Dempfle (2002). "Relative expression software tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR." Nucleic Acids Research 30(9): e36.

Podreka, S., A. Georges, B. Maher and C. J. Limpus (1998). "The environmental contaminant DDE fails to influence the outcome of sexual differentiation in the marine turtle Chelonia mydas." Environmental Health Perspectives 106(4): 185.

Pritchard, P. C. H. (1996). Evolution, phylogeny, and current status. The Biology of Sea Turtles. P. L. Lutz and J. A. Musick. Boca Raton, Florida, CRC Press, LLC. 1: 1- 28.

Ramakers, C., J. M. Ruijter, R. H. L. Deprez and A. F. Moorman (2003). "Assumption- free analysis of quantitative real-time polymerase chain reaction (PCR) data." Neuroscience Letters 339(1): 62-66.

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Stephenson, N. G. (1966). "Effects of temperature on reptilian and other cells." Journal of Embryology and Experimental Morphology 16(3): 455-467. 50

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Storelli, M., G. Barone and G. Marcotrigiano (2007). "Polychlorinated biphenyls and other chlorinated organic contaminants in the tissues of Mediterranean loggerhead turtle Caretta caretta." Science of the Total Environment 373(2): 456-463.

Wallace, B. P., A. D. DiMatteo, A. B. Bolten, M. Y. Chaloupka, B. J. Hutchinson, F. A. Abreu-Grobois, J. A. Mortimer, J. A. Seminoff, D. Amorocho and K. A. Bjorndal (2011). "Global Conservation Priorities for Marine Turtles." PLoS One 6(9): e24510.

Wallace, B. P. and T. T. Jones (2008). "What makes marine turtles go: a review of metabolic rates and their consequences." Journal of Experimental Marine Biology and Ecology 356(1): 8-24.

Wiggins, S. (2011). Identification and Characterization of Reptilian Cytochrome P450 1A: Sequencing, Expression, and Inducibility of CYP1A5 in Sea Turtles. M.S., Texas Tech University.

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of cell number and toxicity." Assay and Drug Development Technologies 3(6): 675-682.

Yamashita, N., K. Kannan, S. Taniyasu, Y. Horii, G. Petrick and T. Gamo (2005). "A global survey of perfluorinated acids in oceans." Marine Pollution Bulletin 51(8): 658-668.

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Chapter 3

Establishment of primary cell cultures from Kemp’s ridley (Lepidochelys kempii) and green (Chelonia mydas) sea turtles

3.1 Introduction Marine turtle populations are declining globally, and along with other reptiles have historically been underrepresented in many fields of research compared to other species (Hopkins 2000, Bolten, Crowder et al. 2010). Cell culture has been used for over a century as a method to investigate biological processes as well as the effects of pharmaceuticals, toxicants, diseases and viruses in animals. The use of immortal cell culture has until recently been more common than primary cell culture in marine turtle research, due to concern with fibropapillomatosis (FP). Fibropapillomatosis is a debilitating disease found in marine turtle species which causes the growth of fibropapillous tumors both externally and on the internal organs (Herbst 1994), and several immortal cell lines have been established from these tumors. The disease is thought to be caused by a virus, and many studies aimed to isolate and/or study this virus using cell cultures from infected turtles (Mansell, Jacobson et al. 1989, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999, Lu, Aguirre et al. 2000, Work, Dagenais et al. 2009). The most common sea turtle species affected by FP is the green sea turtle (Chelonia mydas), and immortal cell lines have been derived several times from this species, either directly from tumors or from unaffected tissues from compromised animals (Koment and Haines 1982, Mansell, Jacobson et al. 1989, Moore, Work et al. 1997, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999). With marine pollution increasing globally, exposure to contaminants and its potential impact on fitness and survival have become more of a concern for marine turtles (Gibbons, Scott et al. 2000, Bolten, Crowder et al. 2010) already affected by many stressors including loss of nesting habitat due to coastal development, etc. Two studies pioneered the use of immortal cell cultures from green sea turtles for in vitro toxicity to assess the effects of heavy metals (Tan, Wang et al. 2010, Wang, Tong et al. 2013) and organochlorines (Keller and McClellan-Green 2004). These studies used cell lines

Texas Tech University, Sarah J. Webb, December 2014 established from one debilitated turtle with fibropapillomatosis (Lu, Nerurkar et al. 1999). It is unknown what, if any, interactions exist between exposure to contaminants and FP. It has been proposed that exposure to chemical pollution could increase the incidence of FP, although this is unconfirmed (Aguirre and Lutz 2004). We assert that it will be important to confirm any toxicological findings obtained with cells derived from animals with FP in case the disease affects cell physiology or sensitivity to contaminants. It will also be important to assess the effects of common marine contaminants both on animals with FP and those without. It is thus critical to establish immortal cell lines from animals both with and without FP. It will also be essential to confirm toxicity studies using primary cells to confirm that the processes leading to cell immortality are not interfering with normal toxic response. Recently, primary cell cultures have been described for four sea turtle species, including the green (Chelonia mydas) (Work, Dagenais et al. 2009), hawksbill (Eretmochelys imbricata) (Fukuda, Kurita et al. 2012, Takeshita, Matsuda et al. 2013), olive ridley (Lepidochelys olivacea) (Fukuda, Katayama et al. 2014), and loggerhead (Caretta caretta) (Webb et al., Chapter 2) sea turtle species. Both hawksbill and loggerhead cell cultures have been used in cytotoxicity testing: the cytotoxic effects of chromium were assessed in cells from one healthy hawksbill sea turtle (Wise, Xie et al. 2014), and loggerhead cell cultures derived from multiple healthy individuals were used in cytotoxicity assays focused on benzo(a)pyrene (B(a)P) and perfluorooctanoic acid (PFOA) exposure (Webb et al., Chapter 2). Here, we describe the culture of primary fibroblast cells from Kemp’s ridley (Lepidochelys kempii) and green sea turtles. The study of marine turtles can be especially challenging because while it is necessary to study and understand the species to provide valuable data for conservation plans, it is also very difficult to effectively study these animals due to the stringent regulations in place to protect them. Research methodologies must be non-lethal and minimally invasive, ruling out traditional methods of toxicological study. Primary cell cultures can be developed from a small (6-8mm) skin biopsy, which has little to no effect on turtles (Bjorndal, Reich et al. 2010). These cell cultures have the potential to be very useful in providing relevant information on the cellular physiology of marine turtles and the effects of contaminants and disease.

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3.2 Methods The culture of primary cells from skin biopsies followed the optimal methods described for the characterization and development of culture conditions for loggerhead cell cultures (Webb et al., Chapter 2). Skin biopsies were collected from stranded animals in rehabilitation at the NOAA/NMFS sea turtle facility in Galveston, Texas under U.S. Fish and Wildlife Service Endangered Species Act Section 10a(1)a Scientific Research Permit TE#676379-5. All research complied with institutional animal care guidelines and U.S. Fish and Wildlife requirements for salvaging and holding sea turtles. Biopsies were taken from the rear flipper and shipped overnight in ice-cold shipping medium (RPMI 1640 cell culture medium (Mediumtech) containing 1.0% penicillin/streptomycin (Mediumtech) and 1.0% Amphotericin (Mediumtech)). Upon receipt, biopsies were placed in tissue buffer (phosphate buffered saline (PBS) (Hyclone) with 1.0% penicillin/streptomycin and 1.0% Amphotericin B) and incubated at 4°C for one hour. All further work was performed under in sterile biological safety cabinet. Tissue was dipped in 70% ethanol three times before being placed in a petri dish containing complete cell culture medium (RPMI 1640, 10% Bovine Calf Serum (Corning), 1.0% penicillin/streptomycin and 1.0% Amphotericin) and minced using a scalpel blade. Minced tissue was then moved to a vented 25 cm2 cell culture flask (Corning) where tissue was spread evenly throughout and medium was removed. These flasks were placed inverted in a Steri-Cult CO2 incubator (Thermo Scientific) at 30°C with 5.0% CO2 for 24 hours. Following 24 hours, complete cell culture medium was added to the flasks, which were then placed right side up in the incubator at the previously described conditions. Three times per week approximately three quarters of the medium was replaced. Upon ~80% confluence, cells were passaged to 75 cm2 vented flasks (Corning) to allow further proliferation. Cells were split from the flasks using 0.5% trypsin (HyClone) subsequent to rinsing with PBS. Trypsinized cells were centrifuged, supernatant was discarded, and cells were resuspended in medium and plated in new flasks. Photos of cells were taken periodically to observe growth patterns and morphology of cells.

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3.3 Results Biopsies from six stranded Kemp’s ridley and thirteen green sea turtles were received, and all biopsies resulted in proliferation for a 100% establishment success rate. Cells proliferated from tissue pieces (Figures 3.1 A & B) within 7-11 days for green sea turtles and 7-14 days for Kemp’s ridley turtles. Cells were morphologically fibroblastic, conforming to the stereotypical fibroblast spindle shape (Figure 3.2 A & B). Kemp’s ridley cells aligned into parallel configurations as population density increased, while green sea turtle cells did not (Figure 3.3 A & B). Contact inhibition occurred when flasks were allowed to reach confluence.

3.4 Discussion Sea turtle species globally are facing serious threats. Of the seven sea turtle species all but one, the flatback sea turtle (Natator depressus) which is currently listed as data deficient, are listed as vulnerable, endangered, or critically endangered (IUCN 2014). The Kemp’s ridley sea turtle is currently the most endangered sea turtle species, listed as critically endangered on the IUCN red list (IUCN 2014). To our knowledge, cells from the Kemp’s ridley sea turtle have never before been successfully cultured. Green sea turtles are currently listed as endangered (IUCN 2014). Both primary and immortal cells have been cultured previously from green sea turtles that were FP positive. In contrast, the cultures we describe here are unique in that they were derived from multiple, healthy individuals exhibiting no signs of FP. There has been much speculation and subsequent research on the effects of the Deepwater Horizon crude oil spill in the Gulf of Mexico on Kemp’s ridley sea turtle populations. Kemp’s ridley sea turtles have a much smaller home range when compared to most other sea turtle species. Their nesting grounds are only located on the beaches surrounding the Gulf of Mexico, with the majority of nesting occurring at Rancho Nuevo, Tamaulipas, Mexico, and in the United States at South Padre Island (Seney and Landry 2008). This means that effects to their nesting grounds could be particularly devastating as compared to other species, which have alternative locations for nesting. To date, little

Texas Tech University, Sarah J. Webb, December 2014 is known about how crude oil contaminants affect sea turtles. The establishment of these cultures provides a method of study that has a negligible effect on the survival of individual animals, as opposed to traditional toxicological study methods such as in vivo dosing studies, while still providing valuable data. Additionally, we believe that these cells can be particularly useful in studies of fibropapillomatosis. This disease is known to affect all sea turtle species (Barragan and Sarti 1994, Herbst 1994, D'Amato and Moraes-Neto 2000, Huerta, Pineda et al. 2000, Williams and Bunkley-Williams 2006) in addition to the green sea turtles in which it was initially reported (Smith and Coates 1938). In vitro study methods using cell culture, both primary and immortal, have been used to study the etiology, infectivity, and effects of the disease in green sea turtle cells (Mansell, Jacobson et al. 1989, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999, Lu, Aguirre et al. 2000, Work, Dagenais et al. 2009). Both green and Kemp’s ridley primary cell cultures can be useful in providing cells for study that are unaltered by FP or immortal growth patterns. It is our belief that these cells will be useful for cytotoxicity testing and may also be effectively applied to other in vitro studies of diseases and sea turtle physiology. Cell cultures have been used extensively in in vitro studies in many species, and confirmed cell culture methods for sea turtles can be particularly valuable by 1) providing a model for physiological studies of endangered sea turtles, and 2) providing relevant and reliable physiological data for use in management and conservation decision making. While these cells have been successfully cultured in our laboratory, we recommend that a full characterization of culture conditions such as what has been done for the loggerhead (Webb et al., Chapter 2) be performed prior to extensive toxicity testing. Conditions such as incubation temperature and medium serum content can have a major impact on the outcome of toxicity testing, and therefore it is imperative to establish well understood, standard culture conditions for toxicity testing (Raz, Fogler et al. 1979, Hahn and Shiu 1983, Hestermann, Stegeman et al. 2002).

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Explant tissue Fibroblast

Fibroblast Explant tissue

Figure 3.1. (A) Fibroblasts proliferating from a Kemp’s ridley sea turtle tissue biopsy 15 days following explant establishment. (B) Fibroblasts proliferating from a green sea turtle tissue biopsy 9 days following explant establishment. Images were captured by deconvolution fluorescence microscopy using a QImaging Go-3 high-resolution digital color microscope camera (100X, scale bar = 20μm).

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3.4 References

Aguirre, A. A. and P. L. Lutz (2004). "Marine turtles as sentinels of ecosystem health: is fibropapillomatosis an indicator?" EcoHealth 1(3): 275-283.

Barragan, A. R. and L. Sarti (1994). "A possible case of fibropapilloma in Kemp’s ridley turtle (Lepidochelys kempii)." Marine Turtle Newsletter 67(28).

Bjorndal, K. A., K. J. Reich and A. B. Bolten (2010). "Effect of repeated tissue sampling on growth rates of juvenile loggerhead turtles Caretta caretta." Diseases of aquatic organisms 88(3): 271.

D'Amato, A. F. and M. Moraes-Neto (2000). "First documentation of fibropapillomas verified by histopathology in Eretmochelys imbricata." Marine Turtle Newsletter 89: 12-13.

Gibbons, J. W., D. E. Scott, T. J. Ryan, K. A. Buhlmann, T. D. Tuberville, B. S. Metts, J. L. Greene, T. Mills, Y. Leiden and S. Poppy (2000). "The Global Decline of

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Reptiles, Déjà Vu Amphibians Reptile species are declining on a global scale. Six significant threats to reptile populations are habitat loss and degradation, introduced invasive species, environmental pollution, disease, unsustainable use, and global climate change." BioScience 50(8): 653-666.

Herbst, L. H. (1994). "Fibropapillomatosis of Marine Turtles." Annual Review of Fish Diseases 4: 389-425.

Hopkins, W. A. (2000). "Reptile toxicology: Challenges and Opportunities on the Last Frontier in Vertebrate Ecotoxicology." Environmental Toxicology and Chemistry 19(10): 2391-2393.

Huerta, P., H. Pineda, A. Aguirre, T. Spraker, L. Sarti and A. Barragan (2000). "First confirmed case of fibropapilloma in a leatherback turtle (Dermochelys coriacea)." Dep Commer, NOAA Tech Memo 193.

IUCN. (2014). "International Union for the Conservation of Nature Red List." Retrieved January 14, 2014, 2014, from http://www.iucnredlist.org/

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Koment, R. W. and H. Haines (1982). "Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle, Chelonia mydas." In Vitro 18(3): 227-232.

Seney, E. E. and J. Landry, André M (2008). "Movements of Kemp’s ridley sea turtles nesting on the upper Texas coast: implications for management." Endangered Species Research 4(1-2): 73-84.

Smith, G. and C. Coates (1938). "Fibro-epithelial Growths of the Skin in Large Marine Turtles, Chelonia mydas (Linnaeus)." Zoologica 23: 93-98.

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Williams, E. H. and L. Bunkley-Williams (2006). "Early fibropapillomas in Hawaii and occurrences in all sea turtle species: the panzootic, associated leeches wide- ranging on sea turtles, and species of study leeches should be identified." Journal of virology 80(9): 4643-4644.

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Chapter 4

Effects of Common Marine Contaminants on Loggerhead Sea Turtle Primary Skin Fibroblasts

4.1 Introduction Sea turtles are long-lived marine reptiles which evolved 220 million years ago, making them one of the most primitive extant group of vertebrates on the planet (Li, Wu et al. 2008). They are charismatic animals which has lead to public support of conservation programs and extensive efforts to aid in their survival for decades. Even with these extensive public support and comprehensive conservation efforts, six of the seven species are still listed on the IUCN red list as threatened, endangered, or critically endangered, with the seventh species, the flatback sea turtle (Natator despressus), listed as data deficient (IUCN 2014). Sea turtles face a variety of challenges, including fishing and shrimping activities which can both directly injure or kill turtles and disrupt important habitat; nesting habitat destruction as coastlines are developed; and long term exposure in the marine environment to toxic chemicals (Gibbons, Scott et al. 2000, Hays 2008, Bolten, Crowder et al. 2010).

Exposure to chemical pollution is complex in sea turtle populations. Because they are both long lived and have an extensive range, they have the potential to bioaccumulate contaminants over many years and from many locations. There is a higher likelihood of exposure to multiple contaminants and long-term exposure than is seen in species which have shorter life spans and high site fidelity. Sea turtles come into contact with a variety of chemical contaminants in the marine environment, including components of crude oil and oil related contaminants, polychlorinated biphenyls (PCBs), perfluorinated compounds (PFCs), heavy metals, and many others (Alam and Brim 2000; Storelli and Marcotrigiano 2003; Keller et al. 2005; Keller et al. 2006).

A challenge when assessing toxicological effects in endangered species is determining a method of study which is both non-lethal and minimally invasive. In the past, studies have used in vivo methods in toxicity studies using endangered sea turtles

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(Hall et al. 1983; Lutcavage et al. 1995), but these types of dosing study are now rarely sanctioned. In vitro methods can be very useful, but must be developed and optimized for use in the study of reptiles. The use of in vitro assays is extensive and well accepted for mammalian studies, especially in the development of pharmaceuticals and for studies on human diseases. Unfortunately, similar studies in reptiles are rare and most commonly used assays have not been fully optimized in reptiles. There have been a few studies which have pioneered the use of cell cultures in toxicity testing in sea turtles. Immortal cell lines derived from a green sea turtle (Chelonia mydas) with fibropapillomatosis have been used to assess the effects of heavy metals and organochlorines (Lu, Nerurkar et al. 1999, Keller and McClellan-Green 2004, Tan, Wang et al. 2010, Wang, Tong et al. 2013). More recently, primary cell cultures have been established from a hawksbill sea turtle (Eretmochelys imbricata) in a study looking at heavy metal cytotoxicity (Wise, Xie et al. 2014), and from loggerhead sea turtles (Caretta caretta) assessing the effect of benzo(a)pyrene (B(a)P) and perfluoroalkanoic acid (PFOA) (Webb et al. 2014).

This work utilized cells derived from multiple healthy loggerheads to optimize several common cytotoxicity assays and provide a preliminary assessment of the effects of marine contaminants. Cytotoxicity assays were divided into three main categories: assays assessing cell viability/cell death; assays assessing sublethal effects including the production of reactive oxygen species and alterations to the cell cycle; and assays assessing biomarker gene expression and enzymatic activity. Culture conditions have been optimized for these cells, and preliminary cytotoxicity testing was performed following exposure to two contaminants: B(a)P, which is a prototypical polycyclic aromatic hydrocarbon (PAH), and PFOA, a common perfluorinated compound (PFC) (Webb et al. 2014). We report here on an extension of that work, including assessment of cytotoxic effects from three major classes of known marine contaminant: PAHs, PFCs, and polychlorinated biphenyls (PCBs).

Three PAHs were assessed, including B(a)P, naphthalene, and phenanthrene. PAHs are known carcinogens and marine contaminants (Conney 1982, Douben 2003). BaP has been found in sea turtle eggs in Florida, the result of maternal transfer to embryos (Alam and Brim 2000). Phenanthrene and naphthalene have been found in sea

Texas Tech University, Sarah J. Webb, December 2014 turtle blood as well as eggs (Alam and Brim 2000, Camacho, Boada et al. 2012, Camacho, Luzardo et al. 2013). Recently, two studies on loggerheads reported that Phenanthrene made up the majority of the total PAH concentration in blood, and was found in over 90% of turtles sampled (Camacho, Boada et al. 2012, Camacho, Luzardo et al. 2013). Effects of PAHs have been reported in freshwater turtles. Embryos from snapping turtles, Chelydra serpentina, and painted turtles, Chrysemys picta, carrying high amounts of alkanes and polycyclic aromatic hydrocarbons (PAHs) show an increased risk of deformities including but not limited to: tail and shell deformations, skull abnormalities, missing limbs, deformed neural tube/spine, and organ abnormalities (Bell, Spotila et al. 2006).

PCBs have been found in sea turtle blood and tissue (Corsolini, Aurigi et al. 2000, Keller, Kucklick et al. 2004). Effects of PCB exposure in other animals include hepatotoxicity, immunotoxicity, developmental abnormalities, reproductive disorders, endocrine disruption, and neurobehavioral effects (Brouwer, Reijnders et al. 1989, Safe 1993, Fox 2001). In sea turtles, effects include changes in white blood cell levels and electrolyte levels, and changes in enzyme levels (Keller, Kucklick et al. 2004). In other turtle species, PCBs have been linked to low hatching success and an increase in hatchling deformities (Bishop, Brooks et al. 1991, Bishop, Ng et al. 1996, Bishop, Ng et al. 1998). PCBs may also act as an endocrine disruptor and affect gonad development in turtles (Bergeron, Crews et al. 1994, de Solla, Bishop et al. 1998, de Solla, Bishop et al. 2002). Two PCBs were used in this work, PCB 77 and PCB 126. PCB 77 has been found in loggerhead sea turtles, as has PCB 126 (Corsolini, Aurigi et al. 2000, Miao, Balazs et al. 2001). Although multiple PCB congeners have been reported in sea turtle tissues, PCB 77 and PCB 126 were chosen for the study in part because they are both among the most toxic and the most abundant reported in sea turtles (Corsolini, Aurigi et al. 2000, Miao, Balazs et al. 2001). Aditionally, PCB77 has been reported as the most important contributor to the toxic equivalent (TEQ) in loggerhead sea turtles (Storelli, Barone et al. 2007), and PCB126 has been described as the most toxic PCB congener for sea turtles (Miao, Balazs et al. 2001). Both are coplanar and known to bind to the aryl hydrocarbon receptor, leading to cytochrome P4501 induction (Okey 1990).

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PFCs have been found in loggerhead sea turtle tissues in varying concentrations depending on geographical location (Keller, Kannan et al. 2005). Body burden of PFCs in sea turtles seems to be dependent upon body size, species, age, and habitat, as PFCs bioaccumulate and occur in higher amounts in older, larger turtles, and in species which are more carnivorous (Keller, Kannan et al. 2005). PFCs are known to cause toxic effects in mammals, including tumor production, mitochondrial alterations, lipid alterations, changes in steroid levels, and peroxisome proliferation (Olsen, Burris et al. 2003, O'Brien and Wallace 2004). For this study, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS) were used as representative PFCs. In loggerhead sea turtles sampled on the east coast of the United States PFOA and PFOS are the dominant compounds, making up 60% and 31% of the total PFC concentration respectively (Keller, Kannan et al. 2005). In juvenile loggerhead sea turtles PFOS represents 60% of the total PFC burden in plasma and serum samples (O’Connell, Arendt et al. 2010). The two compounds behave differently, with PFOS correlating with size, and indirectly age, of turtles, while PFOA does not. PFOS concentrations in plasma tend to be affected by the location of turtles when captured, while PFOA concentrations are not (Keller, Kannan et al. 2005). PFOA and PFOS represent both primary contaminants and degradation products of precursor compounds such as fluorotelomer alcohols and perfluorooctanesulfonyl fluoride (Armitage, MacLeod et al. 2009). Both are C8 PFCs, the form that makes up the highest amount of total global emissions (Armitage, MacLeod et al. 2009).

4.2 Methods Cell Culture Establishment

Skin biopsies were taken from multiple healthy loggerhead sea turtles from the NOAA/NMFS sea turtle facility in Galveston, Texas. All biopsies were taken under permits U.S. Fish and Wildlife Service Endangered Species Act Section 10a(1)a Scientific Research Permit TE-676379-5 and Florida Fish and Wildlife Conservation Commission Marine Turtle Permit #MTP-015. Cell culture establishment followed the

Texas Tech University, Sarah J. Webb, December 2014 methods of Webb et al., 2014. In brief, biopsies were shipped in transport medium on ice, and arrived within 24 hours following collection. Upon arrival, the biopsies were cut into small pieces and placed in a T-25 flask (Corning) in complete cell culture medium in a sterile cell culture incubator at 5% CO2, 30°C. Complete cell culture medium consisted of RPMI 1640 (Mediumtech), 10% bovine calf serum (BCS) (HyClone), 1.0% Penicillin/Streptomycin (Mediumtech), and 1.0% Amphotericin (Mediumtech). When cell cultures reached ~80-90% confluence, they were harvested from T-25 flasks via trypsinization and reseeded in T-75 flasks (Corning) to allow for further proliferation until sufficient cell numbers were available for assays.

Dosing

All contaminants were purchased from Accustandard (≥99% pure) and resuspended to make stock solutions in dimethul sulfoxide (DMSO) as a carrier. Dose concentrations were confirmed following reconstitution via LC/MS and HPLC-UV. Dose ranges were based upon concentrations reported in turtle blood or tissue when available (Corsolini, Aurigi et al. 2000, Keller, Kucklick et al. 2004, Keller, Kannan et al. 2005, Storelli, Barone et al. 2007, O’Connell, Arendt et al. 2010, Camacho, Boada et al. 2012, Keller, Ngai et al. 2012, Camacho, Luzardo et al. 2013), see Tables 4.1- 4.3, and also took into account where possible seawater concentrations and concentrations found in the tissue of other marine animals. Additionally, dose ranges were expanded to allow for comparisons to in vitro work in other species. Dose ranges included: B(a)P – 0.01 μM, 0.1 μM, 1.0 μM, 10 μM, and 100 μM; phenanthrene - 0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; naphthalene - 0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; PCB77 - 0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; PCB126 – 0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; PFOA – 0.05 μM, μM 0.5, μM 5.0, μM 50, and 500 μM; PFOS - 0.01 μM, μM 1.0, μM 10, μM 50, and 200 μM. Cells were dosed for 24, 48, 72, or 96 hours for all assays except the reactive oxygen species (ROS) assay, which, due to rapid formation of ROS, was assessed every 30 minutes for four hours. Reported N for each assay refers to the number of animals from which cells were used.

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MTT assays

MTT assays were optimized for use with our characterized cell cultures. For each animal tested, cells were seeded (5X104 viable cells/well) in TC-treated 96-well plates (Greiner Bio One) and maintained for 48 hours at 30C to allow for cell attachment. Cells were tested for viability using trypan blue exclusion prior to seeding. Cells were then dosed, in triplicate, with the toxicant of interest for 24, 48, 72 or 96 hours. Two controls were used, one with carrier in medium only (0.087% dimethyl sulfoxide, DMSO) and one of medium only. Dosed medium was replaced daily for the duration of the dosing period. After exposure period, medium containing contaminant was removed, 100 µL fresh medium was added to each well, followed by 10 µL of MTT (CalBiochem, prepared as 5 mg/mL in PBS, sterile filtered). Length of time for MTT metabolism was optimized for C. caretta cell cultures by testing 4, 12, and 24 hour time points, and 24 hours was found to be the minimal exposure time to MTT which allowed for measurement of absorbance. Following 24 hour MTT exposure, medium was removed from the plate and replaced with 100 µL DMSO to solubilize the formazan product. The plate was covered to eliminate light exposure, place on a plate rocker for 30 min, and read for absorbance at 570 nm with a reference wavelength of 650 nm using a BioTek Synergy™ 4 Microplate Reader and BioTek Gen5™ software.

LDH Assays

LDH assays were performed using a Lactate Dehydrogenase Cytotoxicity Assay Kit (Cayman Chemical 10008882). Because this assay requires only the medium surrounding cells, it was used in conjunction with MTT assays on dosed cells as previously described. Medium was collected prior to the addition of MTT. Absorbance was read on a BioTek Synergy™ 4 Microplate Reader at 490 nm. Standard curves were plotted using absorbance as a function of LDH concentrations and used to calculate LDH activity in each sample (μU/ml).

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ROS Assay

For reactive oxygen species detection assays, cells were seeded in 96-well plates at 50,000 cells per well. Cells were treated in triplicate with toxicants of interest, a carrier (DMSO) control, or 50 μM tert-butyl hydrogen peroxide (thHP) which serves as a positive control. ROS assays were performed using a kit (Abcam ab113851), which uses 2’,7’-dichlorofluorscein diacetate (DCFDA). DCFDA is oxidized by ROS into 2’,7’- dichlorofluorscein (DCF), which fluoresces. Fluorescence was measured using a BioTek Synergy 4™ microplate readerTM at an excitation wavelength of 495 and an emission wavelength of 529 nm. Fluorescence measurements were taken every 30 minutes for four hours.

Cell Cycle Analysis

For cell cycle analysis, cells were divided into six or seven T-75 flasks, depending on number of doses, with one flask designated for each dose of interest and one flask for the DMSO carrier control. Upon completion of exposure time, cells were harvested and fixed overnight at 4°C in 100% ethanol. Following fixation cells were washed three times with PBS and stained with propidium iodide with RNase A. A BD Accuri C6 flow cytometer (BD Biosciences) was used to analyze cell cycle.

AROD assays

For each animal tested, about 12 million fibroblasts were divided equally between ten or twelve T-75 flasks (two per dose) depending on the number of doses tested and incubated in standard conditions (30°C and 0.5% CO2) for 48 hours to allow cell attachment. Two control flasks were exposed to 0.087% DMSO (carrier) only and treatment flasks were exposed to the toxicant of interest for 72 hours, with medium exchanged for fresh dosed medium daily. Total RNA was isolated for future use in other assays using a NucleoSpin®RNAII kit (Clontech, Foster City, CA) and RNA integrity was evaluated by electrophoresis on 1% agarose gels in 1X MOPS buffer. Following dosing, cells were divided with 1/2 of cells used for RNA isolation and 1/2 of cells being used for AROD analysis.

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To measure the activity of cytochromes P4501A, which are common biomarkers of exposure to polycyclic aromatic hydrocarbons and planar halogenated aromatic hydrocarbons (PHAHs), alkoxy-O-dealkylase (AROD) assays were used. Dosed cells were harvested from T-75 flasks, resuspended in phosphate-buffered saline, and plated in 96-well plates. Plates were then placed in a -20°C freezer for a minimum of 1 hour to disrupt cell membranes. Four substrates were used to determine CYP1A activity, including 7-ethoxyresorufin, 7-methoxyresorufin, 7-pentoxyresorufin, and 7- benzyloxyresorufin. These substrates are oxidized in the presences of CYP1A enzymes to form, resorufin, the final fluorescent product, which was measured using a BioTek Synergy 4™ microplate reader at and excitation wavelength of 530 nm and emission wavelength of 586 nm. Dicoumarol was included in the reactions to inhibit cytoplasmic diaphorase, which can also oxidize alkoxyresorufin. To determine the protein concentration in each well, fluorescamine was added at the conclusion of the assay. Fluorescamine reacts with primary amino groups, producing a fluorescent product which can be used to quantify protein concentration at and excitation wavelength of 400 nm and emission wavelength of 460 nm.

Statistical Analyses

Statistical analysis was performed using R (R Foundation for Statistical Computing, Vienna, Austria). For all assays, one-way analysis of variance (ANOVA) was used to detect whether there were statistically significant differences among treatment groups (p≤0.05), and Tukey’s post-hoc analysis was performed as necessary to determine which groups differed from others.

4.3 Results MTT

MTT Assays were performed on cells dosed with B(a)P, naphthalene, phenanthrene, PCB77, PCB 126, PFOA, and PFOS. Benzo(a)pyrene cytotoxicity was assessed at 0.01 μM, 0.1 μM, 1.0 μM, and 10 μM for 24, 48, 72, and 96 hour exposures

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(n=3 animals). A significant increase in mitochondrial activitywas seen at the highest dose (10 μM) at the 72 hour timepoint (p≤0.01) (Figure 4.1). Phenanthrene (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM) and napthalene (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM) cytotoxicity was assessed at 24, 48, and 72 hours (n=4 animals). No effects weredetected for any timepoint for either toxicant (Figures 4.2 and 4.3). Cytotoxicity was assessed for cells dosed with PCB77 (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; n=5 animals) and PCB126 (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; n=5 animals) for 24, 48, 72, and 96 hours. A significant increase in mitochondrial activity was seen at the 10 μM dose for cells dosed with PCB 77 for 72 hours (p≤0.05) (Figure 4.4) and no significant effects were detected for any time point for PCB 126 (Figure 4.5). Assays were performed incells dosed with PFOA (0.05 μM, μM 0.5, μM 5.0, μM 50, and 500 μM; n=5 animals) and PFOS (0.01 μM, μM 1.0, μM 10, μM 50, and 200 μM; n=5 animals) for 24, 48, 72, and 96 hours. A significant decrease in mitochondrial activity was detected at the 500 μM dose of PFOA for 72 and 96 hours with MTT assays (p≤4.488e-6, p≤0.001) (Figure 4.6), as well as in cells dosed with PFOS at the high dose of 200 μM at 24 and 72 hours (p≤0.05, p≤0.01, n=5 animals) (Figure 4.7).

LDH

Benzo(a)pyrene cytotoxicity (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM) was assessed following 24, 48, 72, and 96 hour exposure using LDH assays (n=5 animals). A significant decrease in LDH release was seen at the highest dose at the 48 and 96 hour timepoints (p≤0.05) (Figure 4.8). Cytotoxicity was assessed for cells dosed with PCB77 (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM, 24 and 48 hours, n=5 animals). No significant changes in LDH levels were detected for PCB77 (Figure 4.9). Lactate dehydrogenase assays were run on cells dosed with perfluorooctanoic acid (PFOA) for 24, 48, 72, and 96 hours (n=5 animals). Significant increase in LDH was seen at the 500 μM dose for 48, 72, and 96 hours (p≤0.01) (Figure 4.10).

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ROS

ROS production was measured every 30 minutes for 4 hours (n=3 animals) following exposure to phenanthrene to determine the best timepoint at which to measure ROS induction. A significant reduction in reactive oxygen species was seen at every timepoint and every dose compared to the control except the 0.001 μM dosed at the 30 minute timepoint (Figure 4.11A & B) (p≤0.05).

At one hour exposure to phenanthrene, significant ROS induction was detected in cells treated with the positive control with much lower variability than was seen at 30 minutes (data not shown), and was thus found to be an appropriate timepoint for future ROS studies for future testing(Figure 4.12). Time points above 1 hour detected less ROS induction, and were excluded for this reason.

Cell Cycle Analysis

Cell cycle analysis was performed for cells dosed with phenanthrene (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM, n=2 animals). No significant change was seen in cell cycle distribution, as analyzed using percentage of the population in S or G2/M phases (p≤ 0.05) (Figure 4.13). Cell cycle effects were analyzed for cells dosed with B(a)P (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM) for 24, 48, 72, and 96 hours, n=3 animals). No effects were seen at any dose for any time point based on percentage of the population in S or G2/M phases (data not shown).

AROD

AROD assays were run for cells dosed with PCB77 (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; n=2 animals) and PCB126 (0.01 μM, 0.1 μM, 1.0 μM, and 10 μM; n=1 animals) for 72 hours. For PCB77 dosed cells, low PROD activity was detected at all doses and no enzymatic activity was observed for MROD, EROD, and BROD (Figure 4.14). Cells dosed with PCB126 exhibited low PROD and BROD enzymatic activities (Figure 4.15). No MROD or EROD enzymatic activity was detected for cells dosed with PCB126.

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Cells dosed with benzo(a)pyrene were analyzed for enzymatic activity using AROD assays following exposure for 24, 48, 72, and 96 hours (n=3 animals). Very low levels (<1.0 picomole/minute/mg protein) of BROD activity were recorded for all time points (Figure 4.16). At 72 and 96 hour exposure, BROD activity only occurred in cells with the highest dose (100 μM). At 24 hours, no PROD activity was seen. At 48, 72, and 96 hour exposures very low levels of PROD activity were detected (<0.5 picomole/minute/mg protein), and as with BROD, at 72 and 96 hour exposure activity only occurred in cells with the highest dose (100 μM) (Figure 4.17). No EROD or MROD activity was seen at any dose or time point for B(a)P dosed cells.

4.4 Discussion This work optimized five assays for use with loggerhead sea turtle primary fibroblasts, and provided a preliminary assessment of the cytotoxic effects of chemicals from three classes of known marine contaminants. Two assays which assess cell viability were optimized, MTT and LDH. The MTT assay detected changes in cell viability in four of the tested compounds, including an increase in viability in cells dosed with B(a)P and PCB77, and a decrease in viability in cells dosed with PFOA and PFOS. A significant decrease in LDH was detected in the medium for cells treated with PCB77 and B(a)P at 10 μM, indicating higher viability in these cell populations than in the control population and confirming MTT results. This decrease in LDH in the medium excluded the possibility that the increase in viability seen in MTT assays was due to an increase in mitochondrial activity, as can occur when cells increase energy production to metabolize exogenous compounds or repair damage. PAHs and PCBs have been shown in other species to induce carcinogenesis and increase cell proliferation (Baird, Hooven et al. 2005, Nebert and Dalton 2006). The increase in cell proliferation detected here may be due to carcinogenic effects, but further testing would have to be done to confirm that in these cells. Additionally, increased proliferation could be confirmed using cell counts at the end of dosing.

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The increase in cell viability detected by MTT and LDH assays in cells exposed to PCB 77 and B(a)P was unexpected, as the majority of studies on mammalian cells exposed to the same concentrations exhibited significant cells death (Endo, Monsees et al. 2003, Sánchez-Alonso, López-Aparicio et al. 2003, Knaapen, Curfs et al. 2007, Lin and Yang 2008, Costa, Catania et al. 2010, Melchini, Catania et al. 2011). A small increase in cell proliferation was reported in mammalian kidney cells exposed to PCB 77 (0.1 μg/mL or 0.34 μM) (Chen, Shen et al. 2010), and in human fibroblasts exposed to PCB 101 (1.0 μg/mL or 3.06 μM) (Hashmi, Khan et al. 2014), both attributed to a possible hormetic response. However, differences between reptilian and mammalian cell physiology are unknown, and further research is needed to investigate whether the increase in cell viability could be due to a hofrmetic response, as has been described in mammals, or carcinogenesis, or yet another process. The lack of significant effects in naphthalene, phenanthrene, and PCB 126 as compared to PCB 77 and B(a)P were also unexpected, but may be due to the differences of structure in the compounds. Compound structures are known to have an effect on carcinogenesis, but more research would have to be done in these fibroblasts to determine the mode of action causing the increase of viability to explain the differences reported here.

A significant decrease in mitochondrial enzymatic activity was detected in cells dosed with 500 μM PFOS and 200 μM PFOS while a significant increase was detected in cells dosed with PCB77 and B(a)P at 10 μM. These results were confirmed by LDH assays, which detected an increase in LDH in the medium for cells dosed with PFOS and PFOA, indicating cell death. Cell death has been reported following in vitro exposure to PFOS (Hu and Hu 2009, Florentin 2011, Buhrke, Kibellus et al. 2013) and PFOA (Shabalina, Panaretakis et al. 1999, Florentin 2011, Buhrke, Kibellus et al. 2013) at doses comparable to those which caused a decrease in cell viability in the loggerhead fibroblasts.

Two assays assessing sublethal toxic effects were tested: detection of reactive oxygen species using DCFDA and cell cycle analysis using flow cytometry. . Reactive oxygen species production was significantly inhibited at all phenanthrene doses compared to the carrier control. The tert-butyl Hydrogen Peroxide (thHP) positive control

Texas Tech University, Sarah J. Webb, December 2014 significantly induced reactive oxygen species production when compared to DMSO control cells, confirming the ability of the assay to detect an increase in ROS in these turtle cells. The decrease in ROS levels in the presences of phenanthrene was not correlated with any other cytotoxic effects in any other assays performed on phenanthrene treated cells, and warrants further investigation to determine the cause of the decrease. The metabolites of phenanthrene have been shown to act as ROS scavengers in vitro in rat cerebellum cells or human leukocytes (Kumagai, Nakajima et al. 1998, Milián, Estellés et al. 2004), which could explain the reduction of ROS in phenanthrene-dosed cells compared to control (DMSO treated) cells.

No significant changes in the cell cycle were detected in our preliminary cell cycle analysis following exposure to benzo(a)pyrene at 24, 48, 72, or 96 hours (n=3) or to phenanthrene for 72 hours (n=2). However, non-significant changes in the cells cycle were observed, including a difference of 13.1% in G2/M phase between control and treated cells exposed to phenanthrene. Cell cycle arrest in G1 and G2/M phases has been reported in cells following exposure to PAHs, due to interaction with the aryl hydrocarbon receptor (Puga, Xia et al. 2002), and an increase in trials (N) is needed to fully assess potential effects in loggerhead skin fibroblasts.

Preliminary assays assessing enzymatic activity of CYP1A detected low levels of PROD in PCB77 treated cells, and low levels of BROD and PROD activity for cells exposed to PCB126 and benzo(a)pyrene, with no EROD or MROD activity seen for any timepoint or dose. Activity was very low (<3 picomole/min/mg protein) for all samples and doses. BROD and PROD activity only showed activity at the highest B(a)P dose (10 μM) at 72 and 96 hour. These data indicate that BROD and PROD may be more relevant than other assays when assessing enzymatic effects in chelonians, and may show more significant enzymatic activity at higher doses of PAHs or PCBs or longer exposure periods. Human fibroblasts have been shown to have repressed CYP1A expression following exposure to dioxin, 3-MC, and B(a)P (Gradin, Wilhelmsson et al. 1993, Haarmann-Stemmann, Bothe et al. 2007), while CYP1A induction has been detected in skin fibroblasts from cetaceans (Godard, Smolowitz et al. 2004, Fossi, Casini et al. 2008). More study is needed to determine CYP1A inducibility in reptilian fibroblasts.

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Cytochrome P450 enzymatic activity has been assessed using AROD assays in the American alligator (A. mississippiensis) (Ertl, Stegeman et al. 1998, Ertl, Alworth et al. 1999) and in two chelonian species: the painted turtle (Chrysemys picta) (Rie, Lendas et al. 2000) and the common snapping turtle (Chelydra serpentina) (Bishop, Ng et al. 1998). Low levels of activity were detected in these taxa, (2-18 pmol/min/mg). EROD assays were used in both chelonian species. In liver microsomes from alligators dosed with 3-MC or phenobarbital (3-4 days)_EROD, MROD, and BROD have been reported to respond to CYP1A inducers. Activity levels up to 60 pmol/min/mg (EROD) were detected in these alligators (Jewell, Cummings et al. 1989, Ertl, Alworth et al. 1999).

This work established the use of primary loggerhead fibroblasts for cytotoxicity testing, and confirmed the effectiveness of MTT, LDH, and ROS assays for assessing effects of marine contaminants on these cells. Cell cycle analysis shows normal cell distribution of fibroblast populations in general, but further testing with additional contaminants, higher doses, and an increase in trials (N) would be valuable to affirm that changes in the cell cycle are detectable in these cells. AROD analysis also requires further testing, but revealed that 7-BR and 7-PR may be better substrates for the detection of enzymatic activity in marine turtles.

In the past, the use of reptiles for toxicity testing has not been included in regulatory decision making. Instead, other organisms have been used as surrogates for reptilian species, such as fish or birds. However, there can be large variations in toxic response to the same doses of contaminants among taxa from the same class or even family level (Brunstrom 1988). This leads to the possibility of underestimating toxic effects in reptiles (Weir, Suski et al. 2010), and we assert that it is necessary to assess effects of contaminants on reptilians using reptilian models to account for physiological differences. This is particularly important in the case of endangered species where the effects of exposure to contaminants could have detrimental effects on an already diminishing population.

The data produced from this work could be confirmed in vivo by toxicity testing either in the species of interest or in a surrogate chelonian species, however, this type of

Texas Tech University, Sarah J. Webb, December 2014 testing is rarely sanctioned for endangered species. This type of testing could possibly be accomplished using stranded and injured turtles which are not candidates for rehabilitation. Future testing could also use a surrogate species, such as the red-earred slider (Trachemys scripta elegans) for both the establishment of cell cultures for in vitro work and for in vivo toxicity testing. This type of study would provide a direct link between in vitro and in vivo findings.

Overall, the use of sea turtle cell culture for in vitro toxicity testing has proved useful for detecting a variety of effects and with further development will be a useful tool in the study of adverse effects of marine contaminants on sea turtles. Marine turtle cell culture has been very useful by providing a system for the study of FP (Moore, Work et al. 1997, Lu, Nerurkar et al. 1999, Lu, Aguirre et al. 2000, Cray, Varella et al. 2001, Work, Dagenais et al. 2009), and these cells can provide a source of marine turtle cells from healthy, FP-negative animals. Further studies using these cells could combine the study of contaminant and fibropapillomatosis to help elucidate any link between pollution and fibropapillomatosis, as has been discussed extensively in the literature (Aguirre, Balazs et al. 1994, Herbst 1994, Herbst and Klein 1995, Miao, Balazs et al. 2001, dos Santos, Martins et al. 2010). The work described here can also be applied toward the development of a model, such as a dynamic energy budget (DEB) model, specific to sea turtles that could be used to extrapolate effects seen in vitro to in vivo effects (Kooijman and Metz 1984, Nisbet, Muller et al. 2000, Kooijman, Baas et al. 2009). The establishment of cell cultures and the confirmation of these assays in sea turtle fibroblasts provide the foundation for the development of this type of model in these species.

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Benzo(a)pyrene mass/mass or mass/volume Molarity Reference(s) Concentration in Other Marine Species 8.8-1173.5 ng/g 34.9 nM - 4.65 μM (Leung, Jefferson et al. 2005) (Neale, Kenny et al. 2005, Concentration Used in Vitro in Other Studies 20-100 μM Richardson and Schlenk 2011)

Phenanthrene mass/mass or mass/volume Molarity Reference(s) Concentration in Water 67 ng/L 0.376 nM (Mitra and Bianchi 2003) (Camacho, Boada et al. 2012, Concentration in Marine Turtles 1.21-4.19 ng/mL 0.376 nM - 6.79 nM Camacho, Luzardo et al. 2013)

Naphthalene mass/mass or mass/volume Molarity Reference(s) (Alam and Brim 2000, Camacho, 0.07 ng/mL-1.94 ng/mL; 0.06 0.546 nM - 15.1 nM; Concentration in Marine Turtles Boada et al. 2012, Camacho, to 0.95 ug/g 0.47 μM - 7.41 μM Luzardo et al. 2013)

Table 4.1. Polycyclic aromatic hydrocarbon concentrations reported in the literature in marine turtle blood and/or tissue, in seawater, and in other marine species on which dose ranges were based.

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PCB 77 mass/mass or Molarity References mass/volume (Corsolini, Aurigi et al. 2000, Concentration in Marine Turtles 0.03 ng/g - 1.0 ng/g 0.103 nM - 3.425 nM Storelli, Barone et al. 2007) (Minh, Nakata et al. 2000, Nakata, Concentration in Other Marine Species 0.04 ng/g - 9.1 ng/g 0.137 nM - 32.2 nM Sakakibara et al. 2002) (Nakata, Sakakibara et al. 2002, Concentration Used in vitro in Other Studies 2.8 pM - 19 μM McKinney, De Guise et al. 2006)

PCB 126 mass/mass or Molarity References mass/volume (Ford, Muir et al. 1993, Reich, Jimenez et al. 1999, Corsolini, Aurigi Concentration in Marine Turtles 0.14 ng/g - 4.4 ng/g 0.42 nM - 13.5 nM et al. 2000, Minh, Nakata et al. 2000, Nakata, Sakakibara et al. 2002) 0.01 ng/g - 1494 Concentration in Other Marine Species 0.03 nM - 4.58 μM (Jiménez, Jiménez et al. 2000) ng/g Concentration Used in vitro in Other Studies 2.8 pM - 34 nM (Nakata, Sakakibara et al. 2002)

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PFOS mass/mass or Molarity Reference(s) mass/volume 0.003 pM - 0.14 Concentration in Water 1.1 pg/L -57700 pg/L (Yamashita, Kannan et al. 2005) nM (O’Connell, Arendt et al. 2010, Concentration in Marine Turtles 1.4 ng/mL - 35.5 ng/mL 3.0 nM - 0.15 μM Keller, Ngai et al. 2012)

Concentration in Other Marine Species 0.33 ng/g -13.9 ng/g 0.8 nM-1.18 μM (Gulkowska, Jiang et al. 2006)

Concentration Used in Vitro in Other Studies 5 μM - 800 μM (Hu and Hu 2009, Florentin 2011)

PFOA mass/mass or Molarity Reference(s) mass/volume 0.036 pM - 0.46 Concentration in Water 15-192,000 pg/L (Yamashita, Kannan et al. 2005) nM 0.493 ng/mL - 8.149 (O’Connell, Arendt et al. 2010, Concentration in Marine Turtles 1.0 nM - 0.02 μM ng/mL Keller, Ngai et al. 2012) Concentration in Other Marine Species 0.27 ng/g - 1.67 ng/g 0.65 nM - 4.0 nM (Gulkowska, Jiang et al. 2006) (Shabalina, Panaretakis et al. Concentration Used in Vitro in Other Studies 5 μM - 2000 μM 1999, Florentin 2011, Buhrke, Kibellus et al. 2013)

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200

150

150 100

100 % Viability % % Viability % 50 50

0 0 0.01 µM 0.1 µM 1 µM 10 µM 0.01 µM 0.1 µM 1 µM 10 µM Benzo(a)pyrene Dose A Benzo(a)pyrene Dose B

180 180

130 ** 130

80 80

% Viability % % Viability % 30 30

-20 0.01 µM 0.1 µM 1 µM 10 µM -20 0.01 µM 0.1 µM 1 µM 10 µM Benzo(a)pyrene Dose D Benzo(a)pyrene Dose C

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300 300

250 250

200 200 150 150

100 100

% Viability % % Viability % 50 50 0 0 0.001 µM 0.01 µM 0.1 µM 1 µM 10 µM 0.001 µM 0.01 µM 0.1 µM 1 µM 10 µM Phenanthrene Dose A Phenanthrene Dose B

300

250

200 150

100 % Viability % 50 0 0.001 µM 0.01 µM 0.1 µM 1 µM 10 µM Phenanthrene Dose C

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160 160

140 140

120 120 100 100 80 80

60 60 % Viability % % Viability % 40 40 20 20 0 0 0.01 µM 0.1 µM 1 µM 10 µM 0.01 µM 0.1 µM 1 µM 10 µM Naphthalene Dose A Naphthalene Dose B

160 140

120 100 80 60 % Viability % 40 20 0 0.01 µM 0.1 µM 1 µM 10 µM Naphthalene Dose C

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250 250

200 200

150 150

100 100

% Viability % % Viability % 50 50

0 0 0.01 µM 0.1 µM 1 µM 10 µM 0.01 µM 0.1 µM 1 µM 10 µM PCB 77 Dose A PCB 77 Dose B

250 250

200

200 * 150 150

100 100

% Viability % % Viability % 50 50

0 0 0.01 µM 0.1 µM 1 µM 10 µM 0.01 µM 0.1 µM 1 µM 10 µM PCB 77 Dose C PCB 77 Dose D

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160 160

140 140

120 120 100 100 80 80

60 60 % Viability % % Viability % 40 40 20 20 0 0 0.01 µM 0.1 µM 1 µM 10 µM 0.01 µM 0.1 µM 1 µM 10 µM PCB 126 Dose PCB 126 Dose B A 160 160 140

140

120 120 100 100 80 80

60 60 % Viability % 40 Viability % 40 20 20 0 0 0.01 µM 0.1 µM 1 µM 10 µM 0.01 µM 0.1 µM 1 µM 10 µM PCB 126 Dose C PCB 126 Dose D

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160 160

140 140

120 120 100 100 80 80

60 60 % Viability % 40 Viability % 40 20 20 0 0 0.05 µM 0.5 µM 5.0 µM 50 µM 500 µM 0.05 µM 0.5 µM 5.0 µM 50 µM 500 µM PFOA Dose A PFOA Dose B

160 160

140 140

120 120 100 100 80 *** 80

60 60 % Viability % 40 Viability % 40 *** 20 20 0 0 0.05 µM 0.5 µM 5.0 µM 50 µM 500 µM 0.05 µM 0.5 µM 5.0 µM 50 µM 500 µM PFOA Dose C PFOA Dose D

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250 250

200 200 150 150

100 100 % Viability % 50 * Viability % 50 0 0 0.1 µM 1 µM 10 µM 50 µM 200 µM 0.1 µM 1 µM 10 µM 50 µM 200 µM PFOA Dose A PFOA Dose B

250 250

200

150 150

100 100

% Viability % % Viability % 50 ** 50

0 0 0.1 µM 1 µM 10 µM 50 µM 200 µM 0.1 µM 1 µM 10 µM 50 µM 200 µM PFOA Dose C PFOA Dose D

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140% 140% 120% 120% 100% 100% *

80% 80% 60% 60%

40% control 40% control 20% 20%

0% 0% % LDH relase relative to to relative relase LDH %

% LDH relase relative to to relative relase LDH % 0.01 µM 0.1 µM 1 µM 10 µM 0.01 µM 0.1 µM 1 µM 10 µM Benzo(a)pyrene dose A Benzo(a)pyrene dose B

140% 140% 120% 120% 100% 100% * 80%

80% 60% 60%

40% control 40% control 20% 20% 0%

% LDH relase relative to to relative relase LDH % 0% 0.01 µM 0.1 µM 1 µM 10 µM % LDH relase relative to to relative relase LDH % 0.01 µM 0.1 µM 1 µM 10 µM Benzo(a)pyrene dose C Benzo(a)pyrene dose D

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140% 140% 120% 120% 100% 100% 80% 80% 60% 60% 40% 40% 20% 20%

0% 0% % LDH relase relative to control to relative relase LDH % 0.01 µM 0.1 µM 1 µM 10 µM control to relative relase LDH % 0.01 µM 0.1 µM 1 µM 10 µM PCB 77 dose A PCB 77 dose B

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250% 250%

200% 200%

** 150% 150%

100% 100%

control control 50% 50%

0% to relative relase LDH % 0% % LDH relase relative to to relative relase LDH % 0.05 μM 0.5 μM 5.0 μM 50 μM 500 μM 0.05 μM 0.5 μM 5.0 μM 50 μM 500 μM PFOA dose A PFOA dose B

*** 250% *** 250% c c 200% 200%

150%

100% 100% control

control 50% 50% 0% 0%

0.05 μM 0.5 μM 5.0 μM 50 μM 500 μM to relative relase LDH % 0.05 μM 0.5 μM 5.0 μM 50 μM 500 μM % LDH relase relative to to relative relase LDH % PFOA dose C PFOA dose D

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800% 700%

600% Pos Control 500% 0.001 uM 400% 0.01 uM 300% 200% 0.1 uM

100% 1.0 uM Percent ROS production production ROS Percent relative to control (DMSO) control to relative 0% 10 uM 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 B Exposure time (hours) A

100%

90% 80% 70% 60% 0.001 uM 50% 0.01 uM 40% 0.1 uM 30% 20% 1.0 uM

Percent ROS production production ROS Percent 10% 10 uM relative to control (DMSO) control to relative 0% 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Exposure time (hours) B

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100%

90%

80%

70%

60% *** 50%

40% *** 30% *** ***

20% *** % ROS Compared ROS % DMSO to Control 10%

0% 0.001 uM 0.01 uM 0.1 uM 1.0 uM 10 uM Phenanthrene Dose (1 hour exposure)

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0.4

0.35

0.3

0.25

0.2

0.15

0.1

pmol resorufin/min/mg protein resorufin/min/mg pmol 0.05

0 DMSO 0.01 µM 0.1 µM 1.0 µM 10 µM PCB 77 Dose

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0.08

0.07

0.06

0.05

0.04 (PROD) 0.03

0.02

0.01 pmol resorufin/minute/mg protein protein resorufin/minute/mg pmol 0 DMSO µM 0.01 µM 0.1 µM 1.0 µM 10 µM PCB 126 Dose A

0.08

0.07

0.06

0.05

0.04 (BROD) 0.03

0.02

0.01 pmol resorufin/minute/mg protein protein resorufin/minute/mg pmol 0 DMSO µM 0.01 µM 0.1 µM 1.0 µM 10 µM PCB 126 Dose B

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0.9

0.8

0.7

0.6 24 h BROD 0.5 48 h BROD (BROD) 0.4 72 h BROD 0.3 96 h BROD 0.2

Picomole resorufin/minute/mg protein protein resorufin/minute/mg Picomole 0.1

0 DMSO 0.01 uM 0.1 uM 1 uM 10 uM 100 uM Benzo(a)pyrene Dose

Figure 4.16. AROD assay following exposure to benzo(a)pyrene in Caretta caretta primary skin fibroblasts, showing BROD activity (means +SEM; n=3 animals).

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0.09

0.08

0.07

0.06

0.05 24 h PROD

0.04 48 h PROD (PROD) 72 h PROD 0.03 96 h PROD 0.02

0.01 Picomole resorufin/minute/mg protein protein resorufin/minute/mg Picomole 0 DMSO 0.01 uM 0.1 uM 1 uM 10 uM 100 uM Benzo(a)pyrene Dose

Figure 4.17. AROD assay following exposure to benzo(a)pyrene in Caretta caretta primary skin fibroblasts, showing PROD activity (means +SEM; n=3 animals).

Texas Tech University, Sarah J. Webb, December 2014

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Neale, J. C., T. P. Kenny, R. S. Tjeerdema and M. E. Gershwin (2005). "PAH-and PCB- induced alterations of protein tyrosine kinase and cytokine gene transcription in harbor seal (Phoca vitulina) PBMC." Clinical and Developmental Immunology 12(2): 91-97.

Nebert, D. W. and T. P. Dalton (2006). "The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis." Nature Reviews Cancer 6(12): 947-960.

Nisbet, R., E. Muller, K. Lika and S. Kooijman (2000). "From molecules to ecosystems through dynamic energy budget models." Journal of animal ecology 69(6): 913- 926.

O'Brien, T. M. and K. B. Wallace (2004). "Mitochondrial permeability transition as the critical target of N-acetyl perfluorooctane sulfonamide toxicity in vitro." Toxicological Sciences 82(1): 333-340.

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Okey, A. B. (1990). "Enzyme induction in the cytochrome P-450 system." Pharmacology & therapeutics 45(2): 241-298.

Olsen, G. W., J. M. Burris, M. M. Burlew and J. H. Mandel (2003). "Epidemiologic assessment of worker serum perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) concentrations and medical surveillance examinations." Journal of Occupational and Environmental Medicine 45(3): 260- 270.

Puga, A., Y. Xia and C. Elferink (2002). "Role of the aryl hydrocarbon receptor in cell cycle regulation." Chemico-biological interactions 141(1): 117-130.

Reich, S., B. Jimenez, L. Marsili, L. M. Hernández, V. Schurig and M. J. González (1999). "Congener specific determination and enantiomeric ratios of chiral polychlorinated biphenyls in striped dolphins (Stenella coeruleoalba) from the Mediterranean Sea." Environmental science & technology 33(11): 1787-1793.

Richardson, K. L. and D. Schlenk (2011). "Biotransformation of 2, 2′, 5, 5′- tetrachlorobiphenyl (PCB 52) and 3, 3′, 4, 4′-tetrachlorobiphenyl (PCB 77) by liver microsomes from four species of sea turtles." Chemical research in toxicology 24(5): 718-725.

Rie, M., K. Lendas, B. Woodin, J. Stegeman and I. Callard (2000). "Hepatic biotransformation enzymes in a sentinel species, the painted turtle (Chrysemys picta), from Cape Cod, Massachusetts: seasonal-, sex-and location related differences." Biomarkers 5(5): 382-394.

Safe, S. (1993). "Toxicology, structure-function relationship, and human and environmental health impacts of polychlorinated biphenyls: progress and problems." Environmental health perspectives 100: 259.

Sánchez-Alonso, J. A., P. López-Aparicio, M. a. N. Recio and M. A. Pérez-Albarsanz (2003). "Apoptosis-mediumted neurotoxic potential of a planar (PCB 77) and a nonplanar (PCB 153) polychlorinated biphenyl congeners in neuronal cell cultures." Toxicology letters 144(3): 337-349.

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Shabalina, I. G., T. Panaretakis, A. Bergstrand and J. W. Depierre (1999). "Effects of the rodent peroxisome proliferator and hepatocarcinogen, perfluorooctanoic acid, on apoptosis in human hepatoma HepG2 cells." Carcinogenesis 20(12): 2237-2246.

Weir, S. M., J. G. Suski and C. J. Salice (2010). "Ecological risk of anthropogenic pollutants to reptiles: Evaluating assumptions of sensitivity and exposure." Environmental Pollution 158(12): 3596-3606.

Yamashita, N., K. Kannan, S. Taniyasu, Y. Horii, G. Petrick and T. Gamo (2005). "A global survey of perfluorinated acids in oceans." Marine Pollution Bulletin 51(8): 658-668.

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Chapter 5

Immortalization of Sea Turtle Fibroblasts Using hTERT

5.1 Introduction There are multiple and complex threats to sea turtle populations, which have contributed to the threatened or endangered status of all but one species (the flatback sea turtle, Natator depressus, currently listed as data deficient) (IUCN 2014). One major concern to the health of sea turtle individuals and populations is exposure to marine contaminants (Gibbons, Scott et al. 2000, Hays 2008, Bolten, Crowder et al. 2010). Due to the endangered status of these animals, methods of study are limited to minimally invasive, non-lethal techniques. One such technique is the use of cell culture as a proxy for the organism in toxicity testing. Cell culture research is commonly used in human medical research and pharmaceutical trials, but is much less developed for reptiles, and sea turtles in particular. There have been multiple immortal cell lines derived from the green sea turtle (Chelonia mydas) (Koment and Haines 1982, Mansell, Jacobson et al. 1989, Moore, Work et al. 1997, Herbst, Sundberg et al. 1998, Lu, Nerurkar et al. 1999). These cell lines have been instrumental in the study of fibropapillomatosis (FP), a debilitating infectious virus which causes the growth of multiple tumors both eternally and internally (Herbst 1994). These cell lines were immortal at the time of establishment because they were collected from timorous tissue. While these cells were useful for the study of FP, they may not accurately reflect the physiological responses of cells from un-infected animals in vitro when used in the study of toxicants. An alternative to the use of immortal cell cultures is the use of primary cell cultures derived from normal tissues. These primary cell cultures may present advantages in cytotoxicity testing because they are unaltered and can be derived from healthy individuals. However, there are also challenges with the use of primary cell cultures: unlike immortal cell lines, which are essentially cancerous and able to proliferate indefinitely without entering senescence, these primary cell populations will eventually age to the point that they are no longer functional and die off if not used for assays. There

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Texas Tech University, Sarah J. Webb, December 2014 is also a need for multiple and relatively frequent tissue sampling events to produce enough primary cells for multiple assays. This is a disadvantage both in the time and effort to collect, ship, and establish the biopsies and in waiting for the biopsies to produce enough cells to use for testing. One option for extending the life of cells is to immortalize them experimentally. The immortal sea turtle cell lines derived from FP-positive green sea turtles, C. mydas, have been successfully used in toxicity assays (Keller and McClellan-Green 2004, Tan, Wang et al. 2010). The goal of this work was to determine the effectiveness of human telomerase reverse transcriptase subunit (hTERT) to immortalize cell line derived from healthy sea turtles, as opposed to tumor cells which have had exposure to the FP virus. This immortalization was attempted with cells from three sea turtle species: loggerhead (Caretta caretta), green (C. mydas) and Kemp’s ridley (Lepidochelys kempii). The most common method of cell immortalization is the use of viruses which inhibit senescence. These are usually DNA tumor viruses, including the Simian virus 40 (SV-40), human papillomavirus (HPV), and Epsein-Barr virus (EBV) (Wright, Pereira- Smith et al. 1989, Yeager and Reddel 1999). These viruses extend the life of cells in a variety of ways. SV-40 produces the large T antigen, which inactivates p53 and retinoblastoma proteins, effectively extending the life of the cells (Manfredi and Prives 1994). Human papillomavirus affects two genes, HPV E6 and HPV E7 (Yeager and Reddel 1999). HPV E6 causes the degradation of the p53 protein and upregulates c-myc expression, and helps to activate telomerase (Klingelhutz, Foster et al. 1996), and HPV E7 affects retinoblastoma proteins, preventing senescence (Boyer, Wazer et al. 1996). EBV is used with lymphocytes and may not actually immortalize the cells, but will extend the population doublings (Tahara, Tokutake et al. 1997). It is also possible to immortalize cells through direct induction of telomerase activity (Yeager and Reddel 1999). This has been accomplished in human fibroblasts by transfecting cells with hTERT, which induces telomerase activity (Bodnar, Ouellette et al. 1998). The shortening of telomeres within a cell leads to senescence (Harley 1991). Without intervention, the cell population will reach a Hayflick limit, which is the number of times a cell population can divide before telomeres become short enough that instability in the DNA occurs (Hayflick 1973). If the Hayflick limit is reached, the cell

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Texas Tech University, Sarah J. Webb, December 2014 population enters senescence and cells will begin to undergo apoptosis (Hayflick and Moorhead 1961, Hayflick 1973). If this shortening of telomeres is arrested, such as by increased hTERT concentrations in the cell, the cells will not reach the Hayflick limit. This was the method chosen to immortalize sea turtle fibroblasts, because the use of hTERT for immortalization has also been effective for fibroblasts derived from humans (Bodnar, Ouellette et al. 1998). Vertebrates have the telomere sequence TTAGGG (Moyzis, Buckingham et al. 1988, Meyne, Ratliff et al. 1989), which has been confirmed specifically in the loggerhead sea turtle (Caretta caretta) (Hatase, Sudo et al. 2008). This identical sequence between species allowed for the use in this work of a commercially produced lentivirus designed to insert an hTERT sequence into cells. Immortalization of cells, either by cancer development or experimentally in the laboratory through transduction can lead to changes in physiology. The use of hTERT allows for immortalization of a cell population through direct induction of telomerase. This allows immortalization without further physiological changes seen in cancerous cells (Morales, Holt et al. 1999).

5.2 Methods Immortalization was attempted on primary fibroblasts from three separate species of sea turtle, the green sea turtle (C. mydas), the loggerhead (C. caretta), and the Kemp’s ridley (L. kempii). All animals used were rehabilitated animals and sampling occurred immediumtely prior to release, after animals were healthy as determined by a veterinarian. Biopsies were collected at the NOAA/NMFS sea turtle facility in Galveston, Texas. Loggerhead skin samples were from captive-reared animals, 2-3 years old. Green and Kemp’s ridley skin samples were from wild animals which were rehabilitated at the facility. All tissue samples were collected under the following permits: U.S. Fish and Wildlife Service Endangered Species Act Section 10a(1)a Scientific Research Permit TE- 676379-5 and Florida Fish and Wildlife Conservation Commission Marine Turtle Permit #MTP-015. All research complied with all institutional animal care guidelines. Primary fibroblast cultures were established following the methods of Webb et al. 2014 (Chapter 2). Biopsies were shipped on ice overnight from Galveston to Texas Tech University in shipping medium (RPMI 1640 medium (Mediumtech), 1.0% penicillin-

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Texas Tech University, Sarah J. Webb, December 2014 streptomycin (Mediumtech), 1.0% Amphotericin B (Mediumtech)). Once received, biopsies were placed in sterile phosphate buffered saline (PBS) (Hyclone) supplemented with 1.0% penicillin-streptomycin) and 1.0% Amphotericin B). Biopsies were then rinsed three times with 70% ethanol, and placed in warm complete cell culture medium (RPMI 1640, 10% bovine calf serum (BCS) (Corning), 1.0% penicillin-streptomycin, 1.0% Amphotericin). Tissue was cut into small (1-2mm) pieces and placed into a vented 25 cm2 cell culture flask (Corning). Medium was then removed and flasks placed in a Steri-

Cult CO2 incubator (Thermo Scientific) at 30°C and 5% CO2 for 24 hours, inverted to aid in tissue attachment to the surface of the flask. After 24 hours fresh complete culture medium was added and flasks were incubated at standard conditions (30°C, 5% CO2). Medium was changed every 2-3 days. Once cells were 70-80% confluent, they were harvested and seeded into 12-well plates at 50,000 cells/well for immortalization. A lentiviral vector carrying the hTERT sequence in supernatant was purchased from Applied Biological Materials for the purpose of immortalization (p-Lenti-hTERT). This supernatant was stored at -80°C until use. Once cells reached 60-70% confluence in 12-well plates the viral supernatant was rapidly warmed in a water bath to 37°C. Medium was removed from the plates and replaced with 1 mL viral supernatant containing 10μg/mL polybrene (ABM). Cell cultures were then incubated at standard conditions (30°C, 5.0% CO2) for a period of 24 hours. After this incubation, supernatant was removed and replaced with complete cell culture medium. Cells were incubated for 72 hours at standard conditions, and then medium was changed for complete culture medium containing 10 μg/mL puromycin (ABM) as a selection marker to select for successfully transected cells. Resistance to puromycin is included in the inserted sequence, and thus only successfully transfected cells survived. If cells numbers were low following selection (<100), Pyrex cloning rings (Fisher) were placed around individual cells to aid in growth. Cells from the same animal were maintained in culture and were not exposed to viral supernatant as a reference to compare growth rates and cell morphology, and to observe and compare senescence.

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5.3 Results Immortalization was attempted for cells from two animals each from the Kemp’s ridley (L. kempii) and green sea turtle (C. mydas), and four animals from the loggerhead (C. caretta) (Table 5.1). Of these, cells from one green sea turtle were successfully transfected with the hTERT virus based upon selection via puromycin. These cells have been passaged nine times to date, and have been in culture for three months. Cells maintained the spindle shape characteristic of fibroblasts (Figure 5.1), but did not arrange into parallel configuration as cultures reached confluence (Figure 5.2). To date, non- immortalized cells from the same green sea turtlefibroblast population from which immortalized cells were derived are still being cultured and have not yet reached senescence. Cells post immortalization exhibited no morphological changes in size or shape compared with primary cells from the same green sea turtle. However, immortal cell culture growth rates are more rapid, requiring only 2-3 days to reach confluence in 25 cm2, compared to the primary cells seeded in the same numbers which take 6-7 days. The green sea turtle from which cells were immortalized, CM-14-186, was found tangled in seaweed upside down on the shore, lethargic and slightly emaciated (Figure 5.3). The sex was undetermined and at the time of collection carapace length was 26.8 cm. The turtle showed no injuries or abnormalities. The turtle was fully rehabilitated and released ~two months later after being cleared for health by a licensed veterinarian. To our knowledge, the turtle was healthy and free of FP at the time of sampling, as the disease does not occur in the green sea turtle population in the area (personal communication, Ben Higgins).

5.4 Discussion At this time the reason transduction has been unsuccessful in loggerhead and Kemp’s ridley cells is unknown. It is possible that a longer incubation period or higher concentration of the virus may be more successful, but further testing is required. It is possible that green sea turtle cells are more susceptible to viral infection, which may explain why that particular species has much higher incidence of the FP virus, while rates are lower in all other turtle species (Herbst 1994, Lackovich, Brown et al. 1999). While this turtle was declared healthy by a veterinarian, and there is no record of FP in the

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Texas Tech University, Sarah J. Webb, December 2014 source population, the FP-negative status of the cells could be confirmed by testing for the presence of a FP-specific viral sequence (Quackenbush, Work et al. 1998, Greenblatt, Quackenbush et al. 2005). The lack of change in cell morphology is promising for the use of these cells in assays, although further testing is needed comparing toxicity testing with primary cells to identical testing with immortalized cells to determine whether any major physiological changes occurred. Further testing will also include determining the location of the hTERT insert in the cell’s genome to rule out the possibility that it was inserted in an area critical to function or an area which would interfere with toxicity testing such as in the middle of a gene coding for biotransformation enzymes. The successful immortalization of these cells is an important development for toxicological study in sea turtles. These green turtle cell cultures provide a tool that has the benefits of both primary cell cultures and immortal cell lines, and few of the challenges. Like primary cell cultures, these cells were derived from a healthy individual. However, there is no need for repeated sampling and extended culture to produce cells for use in assays. These benefits of immortal cell cultures are present without the exposure to fibropapillomatosis that other immortal sea turtle cell cultures have all had. Because the immortalization was induced, the genetic change which induced immortality is known, allowing us to take that into account in any subsequent toxicity studies.

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Species Laboratory ID Source C. caretta CC-13-1692 NOAA/NMFS Galveston, TX Captive Rear Program CC-13-1939 NOAA/NMFS Galveston, TX Captive Rear Program CC-14-097 NOAA/NMFS Galveston, TX Captive Rear Program CC-14-102 NOAA/NMFS Galveston, TX Captive Rear Program C. mydas CM-14-088 Stranded wild turtle, rehabilitated at NOAA/NMFS Facility, Galveston, TX CM-14-186 Stranded wild turtle, rehabilitated at NOAA/NMFS Facility, Galveston, TX L. kempii LK-14-189 Stranded wild turtle, rehabilitated at NOAA/NMFS Facility, Galveston, TX LK-14-191 Stranded wild turtle, rehabilitated at NOAA/NMFS Facility, Galveston, TX

Table 5.1. Animals from which primary skin fibroblasts were exposed to hTERT lentivirus carrier in medium. Cells from the animal in bold were successfully transfected.

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Figure 5.3. The turtle from which cells were successfully immortalized, a stranded and rehabilitated wild green sea turtle (Chelonia mydas).

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5.4 References

Bodnar, A. G., M. Ouellette, M. Frolkis, S. E. Holt, C.-P. Chiu, G. B. Morin, C. B. Harley, J. W. Shay, S. Lichtsteiner and W. E. Wright (1998). "Extension of lifespan by introduction of telomerase into normal human cells." Science 279(5349): 349-352.

Boyer, S. N., D. E. Wazer and V. Band (1996). "E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway." Cancer research 56(20): 4620-4624.

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Harley, C. B. (1991). "Telomere loss: mitotic clock or genetic time bomb?" Mutation Research/DNAging 256(2): 271-282.

Hatase, H., R. Sudo, K. K. Watanabe, T. Kasugai, T. Saito, H. Okamoto, I. Uchida and K. Tsukamoto (2008). "Shorter telomere length with age in the loggerhead turtle: a new hope for live sea turtle age estimation." Genes & genetic systems 83(5): 423- 426.

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Hayflick, L. (1973). Subculturing Human Diploid Fibroblast Cultures. Tissue Culture, Methods and Applications. J. Paul F. Kruse and J. M.K. Patterson. New York, New York, Academic Press: 220.

Hayflick, L. and P. S. Moorhead (1961). "The serial cultivation of human diploid cell strains." Experimental cell research 25(3): 585-621.

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Koment, R. W. and H. Haines (1982). "Characterization of a reptilian epithelioid skin cell line derived from the green sea turtle, Chelonia mydas." In Vitro 18(3): 227-232.

Lackovich, J. K., D. R. Brown, B. L. Homer, R. L. Garber, D. R. Mader, R. H. Moretti, A. D. Patterson, L. H. Herbst, J. Oros and E. R. Jacobson (1999). "Association of Herpesvirus with Fibropapillomatosis of the Green turtle Chelonia mydas and the Loggerhead Turtle Caretta caretta in Florida." Diseases of aquatic organisms 37(2): 89-97.

Lu, Y., V. R. Nerurkar, A. A. Aguirre, T. M. Work, G. H. Balazs and R. Yanagihara (1999). "Establishment and characterization of 13 cell lines from a green turtle

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(Chelonia mydas) with fibropapillomas." In Vitro Cellular & Developmental Biology-Animal 35(7): 389-393.

Manfredi, J. J. and C. Prives (1994). "The transforming activity of simian virus 40 large tumor antigen." Biochimica et Biophysica Acta (BBA)-Reviews on Cancer 1198(1): 65-83.

Meyne, J., R. L. Ratliff and R. K. MoYzIs (1989). "Conservation of the human telomere sequence (TTAGGG) n among vertebrates." Proceedings of the National Academy of Sciences 86(18): 7049-7053.

Morales, C. P., S. E. Holt, M. Ouellette, K. J. Kaur, Y. Yan, K. S. Wilson, M. A. White, W. E. Wright and J. W. Shay (1999). "Absence of cancer–associated changes in human fibroblasts immortalized with telomerase." Nature genetics 21(1): 115- 118.

Moyzis, R. K., J. M. Buckingham, L. S. Cram, M. Dani, L. L. Deaven, M. D. Jones, J. Meyne, R. L. Ratliff and J.-R. Wu (1988). "A highly conserved repetitive DNA sequence,(TTAGGG) n, present at the telomeres of human chromosomes." Proceedings of the National Academy of Sciences 85(18): 6622-6626.

Quackenbush, S. L., T. M. Work, G. H. Balazs, R. N. Casey, J. Rovnak, A. Chaves, L. duToit, J. D. Baines, C. R. Parrish and P. R. Bowser (1998). "Three Closely Related Herpesviruses are Associated with Fibropapillomatosis in Marine Turtles." Virology 246(2): 392-399.

Tahara, H., Y. Tokutake, S. Maeda, H. Kataoka, T. Watanabe, M. Satoh, T. Matsumoto, M. Sugawara, T. Ide and M. Goto (1997). "Abnormal telomere dynamics of B-

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lymphoblastoid cell strains from Werner's syndrome patients transformed by Epstein-Barr virus." Oncogene 15(16): 1911-1920.

Wright, W. E., O. M. Pereira-Smith and J. W. Shay (1989). "Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts." Molecular and cellular biology 9(7): 3088-3092.

Yeager, T. R. and R. R. Reddel (1999). "Constructing immortalized human cell lines." Current opinion in biotechnology 10(5): 465-469.

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