key: cord-0287746-hornbaef
authors: Guillette, T. C.; Jackson, Thomas W.; Guillette, Matthew P.; McCord, James P.; Belcher, Scott M.
title: Blood Concentrations of Per- and Polyfluoroalkyl Substances are Associated with Lupus-like Autoimmunity in the American Alligator
date: 2022-02-19
journal: bioRxiv
DOI: 10.1101/2022.02.15.480575
sha: a47786acc155360f3148388d14ba7a6567926d7c
doc_id: 287746
cord_uid: hornbaef

Surface and ground water of the Cape Fear River basin in central and coastal North Carolina is contaminated with high levels of per- and polyfluoroalkyl substances (PFAS). Elevated levels of PFAS have also been found in blood of fish and wildlife living in and around the Cape Fear River, and in the blood of human populations reliant on contaminated well or surface water from the Cape Fear River basin as a sources of drinking water. While the public and environmental health impacts of long-term PFAS exposures are poorly understood, elevated blood concentrations of some PFAS are linked with immunotoxicity and increased incidence of some chronic autoimmune diseases in human populations. The goal of this study was to evaluate PFAS exposure and biomarkers related to immune health in populations of American alligators (Alligator mississippiensis), a protected and predictive sentinel species of adverse effects caused by persistent toxic pollutants. We found that serum PFAS concentrations in a representative population of alligators from the Cape Fear River were increased compared to a reference population of alligators from the adjoining Lumber River basin. The elevated serum PFAS concentrations in the Cape Fear River alligators were associated with increased innate immune activities and hallmark phenotypes of the human autoimmune disease systemic lupus erythematosus. In addition to significant increases in double stranded-DNA binding autoantibodies in adult Cape Fear River alligators, our qRT-PCR analysis found remarkably high induction of Interferon-α signature genes implicated in the pathology of human autoimmune disease. We interpret the association of increased PFAS exposure with disrupted immune functions to indicate that PFAS broadly alters immune activities resulting in autoimmune disease in American Alligators. This work substantiates and extends evidence from experimental models and human epidemiology studies showing that some PFAS are immune toxicants. Highlights Populations of American Alligator (Alligator mississippiensis) from the Cape Fear River in NC have elevated PFAS exposures compared to a nearby reference population Increased blood PFAS concentrations are associated with increased innate immune activities and hallmark phenotypes of autoimmune disease including increases in autoantibodies Compared the reference population, expression of interferon alpha responsive signature genes that are predictive of lupus erythematosus in humans was increased ∼ 60- to 577- fold in the Cape Fear River Alligators with elevated blood PFAS

• Populations of American Alligator (Alligator mississippiensis) from the Cape Fear River 21 in NC have elevated PFAS exposures compared to a nearby reference population 22

• Increased blood PFAS concentrations are associated with increased innate immune 23 activities and hallmark phenotypes of autoimmune disease including increases in 24 autoantibodies 25

• Compared the reference population, expression of interferon alpha responsive signature 26 genes that are predictive of lupus erythematosus in humans was increased ~ 60-to 577-27 fold in the Cape Fear River Alligators with elevated blood PFAS 28 widespread use over the past 70 years, many PFAS and their terminal breakdown products, have 60 become ubiquitous contaminants of the land, water, and air to which humans and wildlife are 61 exposed (De Silva et al. 2021 Immediately following capture a 10-15 mL a whole blood sample was collected from the 128 post-occipital spinal venous sinus using a sterile 20 g or 18 g needle and a 30 mL syringe 129 (Myburgh et al. 2014) . Whole blood samples were transferred to 8 mL serum and lithium 130 heparin coated plasma tubes (Vacutainer, BD). Serum tubes were allowed to clot for 30 minutes 131 at ambient temperature prior to storage on ice. Plasma tubes were inverted gently following 132 collection, then stored on ice until centrifugation. After field collections, blood was centrifuged 133

(1800 x g for 10 min at 4°C) and serum/plasma fractions were immediately aliquoted into 134

Teflon-free cryovials and stored at -80°C until analysis. Following the blood sample collections, 135 alligators were visually examined for general health, and external injuries were noted and 136 photographed. Sex was determined by cloacal examination with total length, snout to vent 137 length, tail girth, head/snout, and rear foot measurements recorded. Following sample/data 138 collection, alligators were released at the site of capture. Typical time from first contact to 139 release of animal was dependent on animal size with the average time to release being < 15 140 minutes. 1957 (n = 6) was used to test the method for reproducibility and stability (Table S2) . Charcoal 163 stripped fetal bovine serum was also spiked with 5 ng/mL of PFAS standards and used as an 164 internal control for accurate measurement (n = 4). Additional control samples included fetal 165 bovine method serum blanks (n = 7), and purified water field blanks (n = 5) made by injecting 4 166 mL of Milli-Q water into vacutainer tubes during field sampling on 5 different days during the 167 sampling period. 168

All control and experimental samples were extracted and analyzed using methods identical 169 to those detailed in our previous studies (Guillette et al. 2020 ). Briefly, 50 µl of each sample was 170 aliquoted into a 2 mL polypropylene tube to which 100 µl of 0.1 M formic acid and internal 171 standards (12.5 ng) were added. Ice-cold ACN (450 µl) was added to the tube and then vortexed 172 for 3 seconds. Samples were then centrifuged at 12,500 x g for 5 min at room temperature. (Table S4) . single plate at 1:500 dilution and on the same plate using an independent dilution of 1:1000, with 243 each analysis independently replicated. The average intra-assay coefficient of variation for all 244 experimental samples was 5.9 ± 1.4% (1:500) and 4.5% ± 1.1% (1:1000). The average between 245 plate coefficient of variation was 7.0 ± 1.7% (1:500) and 6.0 ± 1.5% (1:1000) . The average 246 Absorbance 450 nM observed for each sample diluted at 1:500 was 2.05 ± 0.02 times greater 247 than samples diluted at 1:1000. 248

Blood smears were prepared from ~10 μl of peripheral whole blood taken from a subset of 250 alligators. Blood smears were prepared in the field and allowed to air dry at room temperature on (Fig. 1A) . From the targeted list of 23 PFAS analyzed (Table S1), we found 14 321 different PFAS, including long and short chain perfluoroalkyl acids (PFAA), perfluoroether 322 acids (PFEA) and the fluorotelomer 6:2 FTS in these alligator blood samples (Table S2, of samples from the CFR (Fig. 1 B-D) . Concentrations and detection frequencies of PFEAs 337 (HFPO-DA, Nafion byproduct-2), PFAS congeners related to upstream production and 6:2 FTS 338 discharge from the Chemours Fayetteville Works facility, were exclusive to, or greatly enriched 339 in the CFR serum samples (Fig. 2C, D; Table S2 ). By contrast the PFAS exposure profiles 340 observed in blood of LW alligators were characterized by the presence of bioaccumalitive six-341 carbon and longer PFAAs (Fig.1D) . However, the PFEAs PMPA (15%), PDO4DA (23%), and 342 PFO5DoDa (19%) were also detectable in some alligator serum samples from LW suggesting 343 that there are low levels of PFEA contamination within the Lumber river basin ecosystem (Fig.  344 1D, Table S2 ). 345

Concentrations of sum total PFAS detected in serum of CFR alligator was increased 346 compared to alligators sampled from LW (Fig. 1E) , two-way ANOVA found a significant effect 347 of location on total serum PFAS concertation (F(1, 71) = 27.3, p < .0001). We did not detect 348 differences in total PFAS concentrations between juveniles/sub-adults (SVL < 90 cm) and adults 349 (F(1, 71) = 0.0018, p = .965) at either site, and there was not an interaction between groups (F(1, 350 71) = 0.989, p = .323). Post hoc analysis indicated that serum concentrations of PFAS were 351 significantly increased in both the juveniles/sub-adult (p = .006) and adult alligators (p <.0001) 352 from the CFR (Fig. 1E) . 353

The American alligator is renowned for having a particularly robust and protective 354 antibacterial and antiviral innate immune system (Merchant and Britton 2006; Finger and Gogal 355 2013) . Initially focusing on biomarkers of innate immune function, we found that anti-microbial 356 lysozyme activity was significantly increased in adult CFR alligators compared to adult alligators 357 from LW ( Fig. 2A) . Those findings were similar to our previous findings that high 358 concentrations of PFAS were associated with elevated lysozyme activity in adult striped bass 359 (Morone saxatilis) from the CFR (Guillette et al. 2020). By contrast, we did not observe a 360 significant difference between sites for lysozyme activities of juvenile/subadult alligators, 361 although at LW the lysozyme activity of sexually immature animals compared to adults from the 362 same location was significantly increased (p < 0.0001; Fig. 2A ). The apparent age-related 363 difference in lysozyme activity at LW was unrelated to differences in PFAS exposure, a 364 difference that is likely related to ecological, population density, or other undefined factors at 365 this location. Because of these site-specific differences in lysozyme activity, associations 366 between total PFAS concentrations and lysozyme activity were evaluated only in sexually 367 mature adult alligators. Using Spearman's ranked correlation to assess the relationship between 368 serum total PFAS concentrations and lysozyme activity in adult alligators from both sites, we 369 observed a positive correlation between serum PFAS and lysozyme activity, r(29) = .45, p = .006 370 (Fig. 2B) . Serum complement activity, a key component of the humoral innate response that has 371 been linked to autoimmunity (Zipfel 2009) However, during our sampling we observed a notably uncharacteristic increase in incidence of 380 skin lesions, slow healing, and infected wounds in ~20% of sampled CFR alligators (Fig. 3A) . 381

Those lesions were considered reminiscent of autoimmune disease related vasculitis in humans 382 (Shanmugam et al. 2017) . Although fresh and healed wounds, including loss of limbs that were 383 likely related to territorial and mating interactions were observed, similarly infected and slow to 384 heal wounds were not observed in alligators sampled from LW. We also observed decreased 385 clotting in some CFR alligators following blood collection; that phenotype was consistent with 386 our findings of modestly decreased numbers of thrombocytes in whole blood cell counts from 387 CFR alligators (Fig. 3B) . Additionally, we found an increased incidence of abnormal red blood 388 cell phenotypes including schistocytes (helmet cells) associated with PFAS exposure in the CFR 389 samples (Fig. 3C, Table S2 ). Our analysis of whole blood cell counts found no evidence for a 390 general increase in chronic stress; no relationships were detected between sample site or PFAS 391 concentrations and leukocyte differentials, or heterophile/lymphocyte ratios. We also measured 392 relative levels of lipid peroxidation products in plasma which also found no evidence of 393 increased impacts related to general systemic mechanisms of cellular injury and stress in the 394 CFR alligators (Fig. S1) . Cxcl10, Mx1, and Oas1 in CFR alligator blood samples (Fig. 3E) . The finding of extremely high 408 induction of Inf-α signature gene expression in the CFR alligators supports a central role for 409 excessive production of type I interferons in increasing the innate immune functions associated 410 with elevated PFAS exposures. We interpret the greatly increased Inf-α signature gene 411 expression, elevated autoantibodies, vasculitis-like skin lesions, elevated incidence of slow 412 healing wounds, and increases in innate immune activity observed in the CFR population as 413 indicative of an SLE autoimmune-like disease process in American alligators. 414

Overall, our work found strong associations across multiple hallmark phenotypes of SLE-like 416 autoimmune disease and elevated PFAS exposures in American alligators exposed to elevated 417 levels of PFAS from the CFR. The PFAS-associated changes in both the innate immune and 418 adaptive immune system may represent a significant health concern for wildlife and human 419 health that may leave exposed populations at greater risk for infections and autoimmune disease. 420

Uniquely, for this work we implemented an integrated One Environmental Health approach 421 using American alligators as sentinels to more fully understand the consequences of long-term 422 exposure to complex and poorly understood mixtures of PFAS with the aim of improving health 423 of impacted human, animals, and ecosystems ( Gibbs 2014; Pérez and Pierce 2018). In addition 424 to being long lived, and sharing their environment with humans, American alligators and other 425 crocodilians, by virtue of having innate immune functions optimized to eliminate microbial 426 pathogens, may serve as an ideal predictive model for detecting autoimmune hazards caused by 427 chemical pollutants. Our findings of adverse immune-health effects in American alligators that 428 have exposure profiles similar to the surrounding human population illuminates and reaffirms the 429 need to reduce exposure and cease production and use of a chemical class that, through its 430 ubiquity and persistence, is a global environmental health concern. 431 Acknowledgments: 432

The authors would like to dedicate this manuscript to the memory of Dr. Louis J. Guillette Jr 433 who continues to inspire us all. 434

We would also like to thank the staff of North Carolina Wildlife Resource Commission, 435 especially Alicia Davis, Christopher Kent, and John Henry Harrelson. We are grateful for the 436 help and tolerance we received from the residents of Wilmington, Lake Waccamaw, Bald Head 437

Island, and Oak Island, NC. We are indebted to our many volunteers, including Deborah and 438

Stephen Burke, Kathy Sykes, Frank Robb, our community partners from the Bald Head Island 439

Conservancy and Cape Fear River Watch who helped with this study, and members of the lab 440 who were not directly involved with this publication. We also thank Dr. Mark Stryner for his 441 analytical assistance, and Dr. Heather Patisaul, Dr. Kylie Rock, and Hannah Starnes for critical 442 review of the manuscript. Although EPA employees contributed to this article, the research 443 presented was not funded by EPA and was not subject to EPA's quality system requirements. 444

Consequently, the views, interpretations, and conclusions expressed in the article are solely those 445 of the authors and do not necessarily reflect or represent EPA's views or policies. 446

Population-Wide Exposure to Per-and 448

Polyfluoroalkyl Substances from Drinking Water in the United States

Perfluorinated Alkyl Acids in Plasma of 453

Alligator Mississippiensis) from Florida and South Carolina

Variation in Perfluoroalkyl Acids in the American Alligator

Merritt Island National Wildlife Refuge

Antimicrobial Peptide from Alligator Mississippiensis Has Antibacterial Activity against 463

Alligators in the Big City: Spatial Ecology of 467

Alligator Mississippiensis) at Multiple Scales across an Urban 468 Landscape

The True 472 Cost of PFAS and the Benefits of Acting Now

Reptiles as Models of Contaminant-Induced Endocrine 475 Disruption

PFAS Exposure Pathways for 482 Humans and Wildlife: A Synthesis of Current Knowledge and Key Gaps in 483 Understanding

Toxicological Effects of Perfluoroalkyl and Polyfluoroalkyl 486 Substances. Molecular and Integrative Toxicology

Per-and Polyfluoroalkyl Substance 490 Toxicity and Human Health Review: Current State of Knowledge and Strategies for 491 Informing Future Research

Endocrine-Disrupting Chemical Exposure and the 494

Immune System of a Top Trophic Carnivore

The Evolution of One Health: A Decade of Progress and Challenges for 498 the Future

Pernille 500 Just Vinholt, Lasse Boding, Carsten Heilmann, and Kåre Mølbak. 2020

Alligators and Endocrine Disrupting Contaminants: A Current Perspective

Elevated Levels of Per-and Polyfluoroalkyl 509 Substances in Cape Fear River Striped Bass (Morone Saxatilis) Are Associated with 510 Biomarkers of Altered Immune and Liver Function

Antimicrobial Peptides in Reptiles

Recently 515 Detected Drinking Water Contaminants: GenX and Other Per-and Polyfluoroalkyl Ether 516 Acids: JOURNAL AWWA

The IUCN Red List of Threatened Species 2019: E.T46583A3009637

Faecal Coliform Bacteria from the American Alligator (Alligator Mississippiensis)

Measurement of Novel, Drinking Water-528

Associated PFAS in Blood from Adults and Children in Wilmington, North Carolina

Identification of Per-and Polyfluoroalkyl Substances 531 in the Cape Fear River by High Resolution Mass Spectrometry and Nontargeted 532 Screening

Saltwater (Crocodylus Porosus) and Freshwater (Crocodylus Johnstoni) Crocodiles

Use 541 for Blood Sample Collection and Intravenous Infusions

Acid (PFOA) and Perfluorooctane Sulfonate (PFOS)

New Comprehensive Global Database of Per-and Polyfluoroalkyl Substances

One Environmental Health: An Emerging 549 Perspective in Toxicology

Tissue-Specific Distribution of Legacy and Novel per-and 553

Polyfluoroalkyl Substances in Juvenile Seabirds

Reconstructing the 557 Composition of Per-and Polyfluoroalkyl Substances in Contemporary Aqueous Film-558 Forming Foams

Vasculitic 561 and Autoimmune Wounds

Rheumatic Diseases

The Ecological Importance of Crocodylians: 568 Towards Evidence-Based Justification for Their Conservation

Diagnostic Hematology of 571 Reptiles

Perfluoroalkyl Substances Are Important Drinking Water Contaminants in the Cape Fear 576

River Watershed of North Carolina

Reptile Hematology

The CompTox Chemistry 582 Dashboard: A Community Data Resource for Environmental Chemistry

Fate of Per-and Polyfluoroalkyl Ether Acids in the Total Oxidizable Precursor 586 Assay and Implications for the Analysis of Impacted Water

Complement and Immune Defense: From Innate Immunity to Human 589 Diseases

Funding: This work was fund by National Institutes of Health grant T32 ES007046 (SMB

National Institutes of Health grant P30 ES025128 (SMB); National Institutes of Health 595 grant P42 ES031009 (SMB)

NC Policy Collaboratory PFAS Testing Network (SMB)

Author contributions: 599 Conceptualization: SMB, TCG, MG 600 Methodology: SMB, TCG, MG, TJ, JM 601 Investigation: SMB, TCG, MG 602 Visualization: SMB, TCG, TJ 603 Funding acquisition: SMB 604 Project administration: SMB 605 Supervision: SMB

Writing -original draft: SMB, TCG 607 Writing -review & editing

All data, code, and materials used in the analysis will be 610 made available for purposes of reproducing or extending the analysis. All data are available 611 in the main text and the supplementary materials