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Inhaling benzene, toluene, nitrogen dioxide and sulfur dioxide, disrupts thyroid function in captive American Kestrels (Falco sparverius) Kim J. Fernie, Luis Cruz-Martinez, Lisa Peters, Vince Palace, and Judit E.G. Smits Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03026 • Publication Date (Web): 19 Sep 2016 Downloaded from http://pubs.acs.org on September 26, 2016

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Environmental Science & Technology

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Inhaling benzene, toluene, nitrogen dioxide and sulfur dioxide, disrupts thyroid function in

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captive American Kestrels (Falco sparverius)

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Kim J. Fernie1*

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Luis Cruz-Martinez2,3

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Lisa Peters4

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Vince Palace5

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Judit E.G. Smits2

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1*

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and Climate Change Canada, 867 Lakeshore Rd., Burlington, Ontario, Canada, L7R 4A6

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2

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Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada, T2N4Z6

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Ross University, School of Veterinarian Medicine, Box 334, Basseterre, St. Kitts, West Indies

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Stantec Consulting Ltd., 500-311 Portage Ave., Winnipeg, MB, Canada R3B 2B9

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IISD-Experimental Lakes Area, 111 Lombard Ave., Suite 325 Winnipeg, MB, Canada R3B 0T4

Ecotoxicology and Wildlife Health Division, Science and Technology Branch, Environment

Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of

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*

Corresponding author: Telephone: +1-905-336-4843; [email protected]

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1 ACS Paragon Plus Environment


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Abstract: Research investigating the effects of air contaminants on biota has been limited to

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date. Captive adult female American kestrels (Falco sparverius) were exposed to a mixture of

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benzene (0.6 ppm), toluene (1 ppm), nitrogen dioxide (NO2; 2 ppm) and sulfur dioxide (SO2; 5.6

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ppm), in a whole-body inhalation chamber. Thyroid axis responses to meet metabolic demands

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were examined through thyroid histology, plasma thyroxine (T4), and triiodothyronine (T3), and

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hepatic outer ring deiodination (T4-ORD). Plasma free (F) T3 and T4 were measured at baseline,

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and at 9-d and 18-d of exposure, while total (T) T3 and TT4, thyroid histology and hepatic T4-

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ORD were determined at the final18-d exposure. Inhalation of these contaminants significantly

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suppressed plasma FT4 and TT4, and depleted follicular colloid and increased epithelial cell

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height at 18-d, and significantly altered the temporal pattern of plasma FT4. Significant

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histological changes in the follicular colloid:epithelial cell height ratio indicated sustained T4

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production and release by the thyroid glands. There was no effect on plasma FT3, TT3, or hepatic

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T4-ORD. We hypothesize that contaminant-related activation of the hypothalamus-pituitary-

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thyroid axis in the kestrels increased elimination of plasma T4 through Phase II enzymes. Further

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research is required to test this hypothesis in wild birds.

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Keywords: air contaminants, oil sands, thyroid hormones, hepatic deiodinase, thyroid gland

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histology, avian sentinels, American kestrels, NO2

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1. Introduction Air pollution consists of multiple chemicals including benzene, toluene, nitrogen dioxide

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(NO2), sulfur dioxide (SO2), and particulate matter (PM). Although these air pollutants have

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generally declined in North America and Europe since 1990, NO2 still exceeds recommended

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standards in some regions and has increased two-fold in Asia, and SO2 emissions have increased

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63% in China, India and many less industrialized regions1. Moreover, in 2013, 87% of people

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globally were exposed to annual average PM2.5 concentrations that exceeded air quality

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guidelines of the World Health Organization1.

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Exposure to air pollutants can cause acute and chronic effects on multiple physiological

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systems and organs, and premature mortality and reduced life expectancy, even from short-term

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exposure2. Exposure to smog and PM increases mortality related to cardiovascular and

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respiratory diseases (e.g., heart disease, lung cancer) as well as non-neoplastic health problems,

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on a global scale3-6. Exposure to even low levels of benzene causes hematological cancers (e.g.,

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7), and adverse effects on reproduction, immune function, the nervous and endocrine systems,

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and cardiovascular and respiratory systems8. Sustained exposure to toluene can cause long-term

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neurobehavioral-endocrine effects and cancer (e.g., 9), while NO2 and SO2 adversely affect the

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immune, blood and nervous systems, and are carcinogenic10,11. Air pollution is generally

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associated with urban centers but also has caused increased disease-related mortality and health

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effects in rural populations1,5. Consequently, wildlife may also be adversely affected by air

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pollution although minimal research has been conducted to date.

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Benzene, toluene, NO2 and SO2 are among the major air pollutants associated with

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industrial mining and related activities in the Athabasca Oil Sands region of Canada12. The

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annual increase of 10% in airborne NO2 concentrations between 2005 and 2010 was considered



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broadly consistent with annual increases in bitumen mining, processing and production13. The

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possible impacts of various chemicals generated by these industrial activities on wildlife and fish

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in this region have been investigated in recent decades. Much of the research has focused on the

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effects of polycyclic aromatic hydrocarbons (PAHs), naphthenic acids, and Oil Sands Processed

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Materials (OSPM) that are released into local wetlands. In birds, changes in reproduction,

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thyroid and immune function, and growth, have been identified in tree swallows (Tachcinyeta

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bicolor)14-16, with the hypothesized causative linkage to exposure to PAHs, naphthenic acids, or

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some other component of OSPM. Exposure to metals and metalloids does not appear to be a

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possible cause of these physiological changes in the birds in light of recent findings indicating

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that their concentrations are similar in tree swallows regardless of their proximity to industrial

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activity in the region17. These birds and other avian species inhabiting the region would have

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been concurrently exposed to benzene, toluene, NO2 and SO2, but the effects of these airborne

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compounds in this region have not been investigated with one exception18.

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Birds have uniquely sensitive respiratory anatomy and physiology19, making them ideal

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candidates to study the effects of air pollutants. In controlled laboratory studies with captive

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American kestrels (Falco sparverius), exposure to benzene, toluene, and in one study NO2 and

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SO2 as well20, elicited hematological changes21, supressed the immune response, reduced plasma

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retinol levels22, and increased corticosterone and hepatic biotransformation activity (7-

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ethoxyresorufin-O-dealkylase (EROD))20.

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Thyroid function is important to the survival of vertebrates because it is involved in

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metabolism, thermoregulation, immune function, growth, and development. Thyroid hormones

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partially govern the timing of reproduction and hence reproductive success, and there is evidence

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to suggest disruption of the thyroid axis among birds exposed to oil sands’ contaminants16. Here,


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we investigated the potential impacts of the inhalation of a mixture of benzene, toluene, NO2 and

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SO2 on the thyroid function of captive adult American kestrels based on maximal concentrations

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measured in the Athabasca Oil Sands region in 201023. In the current study, we hypothesized that

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there would be alterations in thyroid function, which were assessed by measuring circulating free

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(F) and total (T) triiodothyronine (T3) and thyroxine (T4), by examining histological changes

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within the structure of the thyroid gland relating to the production and release of T4, and by

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determining the hepatic outer ring deiodination of T4 to T3 (T4 – ORD).

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Materials and Methods

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Animals and housing conditions

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This study was conducted using protocols approved by the Animal Care Committee of

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the University of Calgary. Captive adult female American kestrels aged 3-7 years (N = 17),

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originally from a long-term breeding colony (the Avian Science & Conservation Center, McGill

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University), were used in this controlled, laboratory study20. None of the birds used were

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experiencing feather molt or approaching their annual breeding season that begins in March-

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April. Each bird was uniquely identified with fluorescent color and aluminum leg bands. The

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kestrels were randomly assigned to one of the two experimental groups (N = 11 exposed kestrels,

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N = 6 controls). The birds were housed together in an indoor flight room (7 m wide x 5 m high x

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3 m long) with controlled ventilation, temperature (21 - 23°C), and light cycle (12-h L:D). The

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flight pen contained two feeding platforms and water baths, and the birds perched on a 1-inch

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diameter rope strung across the room at 1.8 m. The birds were fed an ad libitum diet of 1.5

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frozen/thawed cockerels/kestrel/day soaked in warm water for 30 min prior to feeding each day.

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The kestrels were allowed to acclimate for two weeks before the study began in November 2011.



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Handling stress to the kestrels was minimized by dimming the room lights for cleaning, feeding

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and capturing the birds for blood sampling or transportation to the inhalation chamber.

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Inhalation chamber

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As described in Cruz-Martinez et al.20, a whole body, avian-specific inhalation chamber24

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was modified for this kestrel-inhalation exposure trial: corrosion of the chamber’s components

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was prevented by replacing the copper piping with polytetrafluoroethylene tubing (0.5-inch

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diameter) and the copper fittings with stainless steel fittings (Calgary Valve and Fittings Ltd.,

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Calgary, AB, Canada). The gas mixtures and the hydrocarbon-free air (for controls) were

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released from pre-filled aluminum and steel cylinders (Praxair Inc., Canada, Mississauga, ON,

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Canada; concentration accuracy ± 5%, Manufacturer Guaranteed). All the gas lines merged into

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a 4-piece, stainless steel fitting. Thereafter the gas mixture passed through a flow meter

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(Matheson Tri-gas®, Inc., Basking Ridge, NJ, USA) attached to a bubbler humidifier (Salter

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Labs, Arvin, CA, USA), and then flowed through two stainless steel chambers each with six

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individual animal holding units (straight 2.6-inch diameter PCV tubes with interior perches).

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After passing through the animal holding units, the gas mixture flowed through a carbon-

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activated filter and into the room’s exhaust and scavenger system.

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Gas mixture and rationale for dose selection

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The dose of contaminant gas mixture that the kestrels were exposed to during the course

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of this study was based on air contaminant data recorded during 2010 at 8 (benzene, toluene) and

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9 (NO2, SO2) monitoring stations located throughout the oil sands region in northern Alberta,

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Canada, and managed by the Wood Buffalo Environmental Association (WBEA)23. We used a

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safety factor of 100x the maximal concentrations of these chemicals measured at these WBEA

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stations in 2010, in order to address the variability in the measured responses amongst the


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individual birds, to avoid lethal exposure concentrations, and to permit inter-specific

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extrapolations of the results. Specifically, the 1 h maximal concentrations recorded at each of

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these WBEA stations were averaged, then multiplied by a factor of 100 for benzene and toluene;

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the resulting concentrations were similar to those used in a previous captive kestrel study in

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which there was no evidence of morbidity21,22. There was limited information with which to

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establish non-lethal concentrations of NO2 and SO2, with the closest concentrations being 0.28

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ppm for NO2 and 0.5 ppm SO2 (averaged maximum concentrations recorded in a 6-month period

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near a coal-fired power plant25). Therefore, the 1 h maximum concentrations recorded at each

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WBEA station for NO2 and SO2 were averaged and multiplied by a factor of 100, based on a

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previous study involving quail that involved a factor of 1020. Consequently, the kestrels in the

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control group were exposed to medical grade air, while those in the contaminant group were

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exposed to a dose of 0.6 ppm benzene, 1 ppm toluene, 2 ppm NO2 and 5.6 ppm SO2.

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Dosing protocol and exposure times

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The birds were transported from their flight room to the inhalation chamber where the

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control group was exposed first followed by the contaminant-exposed group. Details of the

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experimental set-up have been described elsewhere20,24. Briefly, the chamber was flushed for 5-7

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mins with hydrocarbon-free air prior to dosing. When the birds were perched quietly inside the

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chamber, the lids of the individual holding units were tightly closed and the gas cylinders were

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opened with a total controlled flow of 10 liters per min (L/min) The flow rate was calculated

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using avian specific air requirements19 adapted to the specific requirements of the kestrels. The

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minimum flow rate suitable for kestrels is 3.5 L/min in order to maintain normal body

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temperature21. As a safety precaution to prevent over-heating of the birds in these small



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chambers, these flow rates were increased to 10 L/min26. The birds remained quietly perched in

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the individual chambers throughout the exposure periods.

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The chamber was tested for potential leaks each day of the trials by applying a non-

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corrosive liquid (Snoop® Liquid Leak Detector, Swagelok, Calgary, AB, Canada) to each joint.

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The official exposure time started once equilibrium was reached inside the chamber,

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approximately 20 min after the gas lines were opened. The equilibrium (T99), the chamber’s

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volume (47.3 liters), and the required flow rate, were based on previous studies24,27. The kestrels

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were exposed for 1.4 hr/day, 5 d/wk for a total of 18 days of exposure since logistical constraints

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necessitated modifying the 4 wk period originally planned. This 18 d period is representative of

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the nestling period of many avian species (e.g., tree swallows) that nest in the oil sands region.

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Sample collection

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Blood samples were collected at the beginning, middle (9 d) and end (18 d) of the

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inhalation exposure trial, by jugular venipuncture using lithium-heparinized 1 ml syringes and

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0.5-inch needles (27 g) at a volume of ≤ 1% of their body weight. The whole blood was kept on

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ice until all samples were collected, then immediately centrifuged and the blood plasma

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separated into aliquots, stored in liquid nitrogen, and then at -80oC until analysis. Blood plasma

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samples were used to determine circulating concentrations of FT3 and FT4 at 0, 9 and 18 d of

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exposure, and TT3 and TT4 at 18 d of exposure only.

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All of the contaminant-exposed kestrels (N = 11), and five of the 6 kestrels in the control

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group, were euthanized at the end of the exposure trial (day 18). The birds were humanely

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euthanized, initially anesthetizing them with inhalation isoflurane using an anesthetic machine,

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before cervical dislocation. A complete necropsy was immediately performed. The liver was

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excised, weighed, dissected and stored in cryovials in liquid nitrogen. The thyroid glands of the


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kestrels were excised, weighed, trimmed consistently and fixed in 10% buffered formalin. All

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samples were relabeled to ensure a blind assessment.

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Analysis of Thyroid Function

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Thyroid function was assessed by determining circulating T3 and T4 concentrations in the

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plasma, hepatic T4-outer ring deiodinase activity (T4-ORD), and histological assessments of the

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thyroid glands. Circulating concentrations of FT3, TT3, FT4, and TT4, determined with modified,

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validated protocols at the National Wildlife Research Center laboratory (NWRC), were analyzed

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in duplicate (> 60% of samples) using 50% of the recommended volume of plasma because the

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small size of the birds prohibited the non-lethal collection of greater blood volumes during the

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course of the experiment. Subsequently, the manufacturer’s instructions were followed using a

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commercially available radioimmunoassay kit (Siemens Medical Solutions Diagnostics RIA

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Coat-a-Count Kits, Inter-Medico (Montreal, QC)), and based on linear response versus

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concentration calibration curves (FT3, TT3, FT4, FT4: R2 > 0.99) across the physiological range of

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hormone concentrations (FT3: 0.22 – 19 pg/ml; TT3: 0.1 – 6.0 ng/ml; FT4: 0.45 – 56 pg/ml; TT4:

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2.4 – 145 ng/ml). The assay detection limits were 0.2 and 0.1pg/ml for FT3 and FT4 respectively,

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and 0.07 and 1.0 ng/ml for TT3 and TT4 respectively. The coefficients of variation were < 20%.

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All modifications were validated, including for species-specificity using pooled plasma that was

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quality assured and quality controlled.

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For the determination of hepatic deiodinase activity, a 100-200 mg sample of each liver

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was homogenized in ice cold 0.1 M sodium phosphate buffer (pH = 7.4) containing 1 mM

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ethylenediaminetetraacetic acid (EDTA) and 20 mM dithiothrietol (DTT), and centrifuged at

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9,000 X g at 4 ºC for 15 minutes. The resulting S9 supernatant was recovered, frozen and stored

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at -80ºC until analysis. To determine deiodinase activity, 10 µl of S9 homogenates were



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incubated at 37ºC in 900 µl of sodium phosphate buffer. At timed intervals, 5 µl of

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dimethylsulfoxide (DMSO) containing 3000 ng of T4 were added to each sample to a maximum

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of < 5%, in order to begin the reactions28. Reactions were terminated 90 minutes later by adding

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1 ml of ice cold methanol. Two types of blanks were included with each analytical run: one with

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all assay constituents except S9, and another containing all constituents except T4. T3 produced

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by the reactions was determined using 100 µl of the final reaction volume tested with a

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commercial radioimmunoassay kit (MP Biomedical, Santa Ana CA, USA). T3 is the most

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relevant and biologically active end product of deiodinase activity. Protein concentration was

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determined in a separate aliquot of S9 homogenate using the Bradford29 method. Deiodinase

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activity was expressed as pmol of T3 liberated per minute per mg of protein, with blank

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correction included as previously described for American kestrels30,31.

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Formalin-fixed tissues were processed and embedded in paraffin using previously

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described procedures32. Tissues were sectioned at approximately 7 µm, mounted on slides, and

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stained with hematoxylin and eosin (H & E). Each histological slide was examined using a Carl

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Zeiss EMS stereoscope with an APoLumar SI.2Y objective, and an AxioCam HRC digital

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camera at 26 milliseconds (ms) exposure to capture images of the thyroid tissues for

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measurement. Digital images of two randomly selected fields of view for each slide were taken

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at 40x magnification. For each thyroid gland, ten complete follicles per image (total of 20

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follicles) were selected and analyzed as described by Park and his colleagues33. Briefly, epithelial

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cell heights (ECH) were measured (µm) at 4 locations per follicle, approximately 90 degrees

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from each other, and then averaged to calculate a mean ECH per follicle. Area (µm2) of the

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colloid inside the same 20 thyroid follicles was also measured. The data were expressed as mean

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thyroid ECH and mean colloid area per individual bird. The mean ratio of colloid


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perimeter:ECH was also calculated as an indicator of thyroid gland activation and the potential

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for thyroid hormone production34. All analyses were performed using Zen Lite 2012 (2012)

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software.

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Statistical Analysis

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The data and residuals for each parameter were tested for normality of distribution and

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homogeneity of variance. Those data that failed these tests were transformed when possible to

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permit parametric tests; otherwise, non-parametric statistical methods were used. Possible

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differences between the two treatment groups in circulating TT3 and TT4 concentrations

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(determined on d 18 only) were assessed using the Kruskal-Wallis test. Possible differences

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between the two groups of birds in body mass, and circulating concentrations of FT3 and FT4,

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were determined using repeated measures analysis of variance (RM ANOVA) with subsequent

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multivariate ANOVA results reported for overall treatment-related effects; the assumption of

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sphericity was met for body mass and plasma FT3 data (p-values ≥ 0.09) but not the FT4 data

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(Mauchly’s Sphericity Criterion; X2 = 13.64 df = 2 p = 0.001); consequently, the degrees of

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freedom were adjusted for the FT4 statistical results. The one-way ANOVAs associated with the

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RM ANOVA analysis were used to identify all treatment-related differences in circulating FT3 or

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FT4 at the individual sampling periods, specifically at baseline (0-d), 9-d and 18-d of exposure.

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Nested ANOVAs on ranked data were used to account for the multiple measurements recorded

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for each glandular histological endpoint on each bird. A Mann-Whitney U-test was used to

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determine changes in hepatic T4-ORD activity resulting from the kestrels inhaling these specific

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gases over the 18-d exposure period. All statistical analyses were conducted using SAS 9.4® and

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significance was set at a p-value (α) of ≤ 0.05.

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Results The inhalation exposure trial had no significant effect on the body mass of the kestrels

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overall (RM ANOVA treatment, treatment*time: p-values ≥ 0.41), or specifically at the

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beginning (0 d) and end (18 d) of the inhalation study (p-values ≥ 0.69).

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Inhaling this combination of benzene, toluene, NO2, and SO2, significantly affected the

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temporal pattern of circulating FT4 concentrations in the kestrels (RM ANOVA treatment*time:

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F1.06,16.93 = 4.38 p = 0.04), with plasma FT4 levels being significantly lower in the exposed

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females than the control female kestrels on d 18 only (F1,10 = 6.78 p = 0.02) (Fig. 1) (Table 1). At

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18 d of exposure, there was a significant reduction in circulating TT4 concentrations in the

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exposed kestrels compared to the controls (X2 df = 1 p = 0.003) (Table 1).

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The FT3 concentrations of the kestrels varied significantly over time (RM ANOVA time:

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F2,10 = 15.67 p = 0.0008), but not as a function of the inhaled contaminants overall or at any of

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the three sampling points (0, 9 and 18 d of exposure) (RM ANOVA treatment, treatment*time: p

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– values ≥ 0.59). Similarly, there was no significant effect of the inhalation treatment on the

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circulating TT3 concentrations at 18-d of exposure (p = 0.44) (Table 1).

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Fig. 1. Circulating free thyroxine (FT4) (ng/mL) in American kestrels during an 18 d trial in

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which the birds inhaled a mixture of benzene, toluene, NO2 and SO2. Inhaling these chemicals

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significantly affected the kestrels’ temporal pattern of circulating FT4, and their plasma FT4

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concentrations at 18-d of exposure (all p-values ≤ 03). Post-hoc least square means and standard

265

errors about the means (SEMs) are presented.


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3.0 Treatment Controls

FT4 (ng/mL)

2.5

2.0

1.5

* p = 0.02 1.0

0.5 0

9

18

Day of Exposure (d)

266 267

Table 1. Comparisons of circulating thyroid hormones measured in adult female American

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kestrels between control birds and birds inhaling a mixture of benzene, toluene, NO2 and SO2.

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Arithmetic means (mean) and standard errors about the means (SEM), as well as the range of

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concentrations of each hormone, are reported. The p-values identify statistically significant

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differences in concentrations between the two groups at each sampling time point as described in

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the Results; N.S. denotes non-significant p-values > 0.05.

FT4

FT3

TT4 TT3

Day of pExposure value 0 N.S. 9 18 0 9 18 18 18

N.S. 0.02 N.S. N.S. N.S. 0.003 N.S.

Control Birds N Mean SEM Range

N

Exposed Birds Mean SEM

4

1.7

0.2

1.3 - 2.2

8

2.7

0.4

0.9 - 4.4

6 6 4 6 6 6 4

1.1 2.4 2.3 1.3 2.1 8.4 1.7

0.2 0.4 0.4 0.1 0.2 1.1 0.1

0.4 - 2.0 1.5 - 3.6 1.2 - 3.3 1.0 - 1.6 1.6 - 2.9 5.6 - 13.0 1.4 - 2.1

9 10 9 9 10 11 7

1.1 1.6 2.0 1.1 2.4 3.5 2.6

0.2 0.2 0.3 0.1 0.2 0.7 0.8

0.2 - 1.9 0.9 - 2.8 1.3 - 3.7 0.1 - 2.6 0.9 - 2.8 0.5 - 7.2 1.3 - 2.5

Range



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Concurrent with the decrease in plasma FT4 in the contaminant-exposed kestrels, there

275

were significant structural changes in the thyroid glands of the same birds. In the exposed

276

kestrels there was a significant decrease in the colloid area of the follicles (nested ANOVA on

277

ranks: F13,293 = 7.40 p < 0.0001) indicating that thyroglobulin was reduced within the thyroid

278

gland (Fig. 2A). This reduction in the precursor thyroglobulin was likely due to increased

279

demand for T4. This suggestion is corroborated by the greater epithelial cell heights (nested

280

ANOVA on Ranks: F13,293 = 6.53 p < 0.0001) indicating activation of the thyroid glands in the

281

exposed birds, since the increased epithelial cell height was concurrent with the reduced colloid

282

available in the gland (Fig. 2B) (nested ANOVA on Ranks: F13,293 = 5.86 p < 0.0001). Together

283

the larger epithelial cells and decreased colloid within the glands suggest that the sustained

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contaminant exposure increased activation of the thyroid glands provoking greater T4 production

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and release into circulation.

286 287

Fig. 2. Histological changes in the thyroid glands of American kestrels exposed for 18 d to

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inhaled benzene, toluene, NO2 and SO2. Exposed kestrels had more activated thyroid glands

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indicated by significantly less colloid area in the follicles (A) and significantly increased

290

epithelial cell height (B) (***p-values < 0.0001). Post-hoc least square means and standard

291

errors about the means (SEMs) are presented.


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3000

A.

2 Colloid Area (um )

2500

2000

***

1500

1000

500

0 Control

292

Colloid Area:Epithelial Cell Height

14

Exposed

B.

12

***

10 8 6 4 2 0 Control

293

Exposed

294 295

There was no significant concurrent change in hepatic T4-ORD activity (Mann-Whitney

296

U-test: 18.00 p = 0.391) at 18-d exposure, indicating that inhalation of this mixture of

297

contaminants at these concentrations did not result in increased conversion of T4 to T3 in the liver

298

of the contaminant exposed birds.

299



300 301

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Discussion The current study with captive kestrels was conducted under controlled, laboratory

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conditions stimulated in part because of earlier studies with wild birds in the oil sands region of

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northern Alberta. Gentes and her colleagues16 reported that there were increased concentrations

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of plasma T3 and glandular T4 in wild nestling tree swallows raised near wetlands partially

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comprised of mine tailings. The authors concluded that T4 production was increased within the

306

thyroid glands of these swallow nestlings, independent of the pituitary-thyroid axis, and

307

hypothesized that there was increased deiodination of T4 to T3 outside of the thyroid gland. Since

308

these were wild swallow nestlings, the mechanisms eliciting such changes could not be identified

309

and the authors suggested exposure to PAHs and/or naphthenic acids, but in combination with

310

environmental factors such as temperature variations and food availability16. The possible role of

311

benzene, toluene, SO2 and NO2, in eliciting these changes in thyroid function was not

312

examined16 although the swallows that they studied would likely have been exposed to these

313

compounds concurrently since they occur in the region12,13.

314

The results of the current study indicate that this subchronic exposure of captive kestrels

315

to benzene, toluene, SO2 and NO2, had significant effects on several components of the avian

316

thyroid system. Significant suppression of FT4 and TT4 at the end of the exposure period (18 d)

317

concurrent with indices of activated thyroid gland activity (i.e., reduced colloid and greater

318

epithelial cell height), suggest that exposure to inhaled contaminants may have removed negative

319

feedback that constitutively inhibits the thyroid gland. However, there were no changes in FT3 at

320

any time, or in circulating concentrations of TT3 at the end of this study. T3 is the primary

321

feedback hormone for negative feedback inhibition of the avian thyroid axis35. The hepatic ORD

322

enzyme activity would be expected to convert T4 secreted by the thyroid gland, but was not


Page 17 of 29


323

significantly affected in the liver which is the major site of conversion. While this deiodinase

324

conversion of T4 also occurs in the kidney, intestine and central nervous system in birds36, as

325

with mammals, hepatic Type I deiodinase (5ꞌD I) is responsible for producing the majority of T3

326

in birds35.

327

The constitutive androstane receptor (CAR) is predominantly found in the liver and small

328

intestine of mammals. Benzene is a potent inducer of CAR and studies have demonstrated that

329

the activation of CAR disrupts thyroid hormone homeostasis37. Closely related to mammalian

330

CAR, the chicken xenobiotic-sensing orphan nuclear receptor (CXR) occurs in birds. Activation

331

of CAR regulates several cytochrome P450s, sulfotransferases, UDP-glucuronosyltransferases,

332

and glutathione transferases38, while CXR regulates hepatic CYP and uridine diphosphate

333

glucuronosyl transferase (UDPGT) enzymes in birds39 and references therein. T4 is degraded mostly by

334

hepatic deiodination (80%) but also by UDPGT, an enzyme that is induced by contaminants

335

(e.g., PCBs, phenochlor) in rats and birds, subsequently enhancing the glucuronidation and

336

excretion of T435 and references therein. The cytochrome P450 enzymes, CYP2E1 and CYP2F2, are the

337

major contributors to the bioactivation of benzene in the lungs of rats and mice40, whereas

338

CYP2E1 also binds to a high proportion of inhaled toluene22 and references therein. CYP2E1 is more

339

readily induced in females and female birds were used in our current study. Avian lungs have

340

greater biotransformation capacity than mammalian lungs19,22. We suggest that T4 may have been

341

eliminated preferentially through activation of the CXR increasing CYP2E1 and UDPGT activity

342

in response to metabolism of the benzene, toluene, and possibly NO2 and SO2 by the kestrels.

343

Hepatic EROD biotransformation was stimulated in the same kestrels20, which has also been

344

observed in wild tree swallow nestlings studied in the Oil Sands region14 including those

345

experiencing thyroid-related changes15,16.



346

Page 18 of 29

In contrast to the study with wild birds by Gentes and colleagues16, the current study was

347

conducted under controlled laboratory conditions with the kestrels having an ad libitum diet,

348

experiencing warm, temperature-controlled conditions, and showing no change in immune

349

function20 – factors that can influence thyroid function in vertebrates35,41. Foraging activity and

350

food consumption can also influence circulating thyroid hormones in vertebrates, but given the

351

indirect evidence of food consumption, with the kestrels in the two groups having similar body

352

mass, we suspect that food intake was not notably influenced because of the inhalation of these

353

chemicals. Consequently, the observed changes seen in circulating FT4, TT4 and glandular

354

histology in the present study were most likely related to the kestrels inhaling the benzene,

355

toluene, SO2 and NO2. We hypothesize that with the exposure to these compounds detoxification

356

demands were increased, disrupting endocrine and physiological pathways within the birds. We

357

further hypothesize that the inhalation of SO2 in particular by the kestrels, may explain i) the

358

reductions in their circulating FT4 and TT4 concentrations, ii) the decrease in glandular colloid

359

and (likely) thyroglobulin, and iii) the increased activation of the thyroid glands since sulfur

360

inhibits iodide transport and organification, the biochemical process in which iodine is

361

incorporated into thyroglobulin during the production of T4 in the thyroid gland42. Further

362

research would be required to test these hypotheses. The observed thyroid responses in the birds

363

are also noteworthy in that they occurred in the captive kestrels after slightly less than 3 weeks of

364

exposure, whereas many species of wild birds breed over the course of 6 weeks or more, and so

365

changes in thyroid responses may be expected, given these longer exposure periods.

366

Free T4 is not protein bound, distinguishing it from the balance of T4 that makes up the

367

total measurable TT4. Consequently, the free form of the hormone is responsible for regulating

368

thyroid gland activation by the pituitary gland, through negative feedback. When circulating FT4


Page 19 of 29


369

levels decline sufficiently, the pituitary-thyroid axis is activated to release thyroid-stimulating

370

hormone (TSH) from the anterior pituitary, which in turn stimulates T4 production by the

371

follicular epithelial cells of the thyroid gland. In wild tree swallows nesting near wetlands

372

receiving OSPM, Gentes and her colleagues16 hypothesized that the increased glandular hormone

373

content without concurrent changes in plasma T4 concentrations, indicated a change in glandular

374

T4 production but not release. Furthermore, since there was no indication of glandular

375

hypertrophy or changes in circulating T4 in the wild swallow nestlings, they concluded that there

376

was no sustained activation of the hypothalamus-pituitary-thyroid axis16 since increased T4

377

production reflects greater activation of the pituitary-thyroid axis in response to declining

378

circulating thyroid hormones. In contrast, for the kestrels in the current controlled laboratory

379

study, sub-chronic inhalation of benzene, toluene, SO2 and NO2 appears to have led to decreased

380

circulating FT4 and TT4 concentrations and sustained activation of the thyroid gland to produce

381

and release glandular T4. This idea is supported by the findings of reduced follicular colloid and

382

larger epithelial cells. Consequently, we hypothesize that sub-chronic contaminant exposure

383

caused activation of the hypothalamus-pituitary-thyroid axis in the kestrels, and subsequent

384

production of TSH to stimulate glandular T4 production and release. Moreover, we hypothesize

385

that at least one of the four chemicals, possibly SO2, inhaled by the birds may have been

386

competing with T4 for the thyroxine transport protein, transthyretin, as occurred in rats exposed

387

to airborne particulate matter43. Further investigation of this mechanism, including determining

388

TSH concentrations, and histology of the hypothalamus and/or the pituitary gland, might offer

389

more insight into the costs of inhaled benzene, toluene, SO2 and NO2.

390 391

In addition to deiodinase enzyme activity, metabolic turnover of thyroid hormones can also affect circulating concentrations. Cellular thyroid receptors bind to T3 with much greater



Page 20 of 29

392

affinity than T436, T3 having greater biological activity than T435. Consequently, T3 may be more

393

tightly controlled physiologically and less subject to elimination than T4, possibly providing an

394

explanation of why only plasma T4 levels were suppressed without any changes in FT3 and TT3

395

in the current kestrels. Consequently, it is not unreasonable to hypothesize that T4 was

396

indiscriminately cleared through Phase II conjugation enzymes because of the upregulation of

397

these detoxification enzymes stimulated by the contaminant exposure. This hypothesis warrants

398

further research.

399

Appropriate thyroid function is essential for the survival of the individual, as it is

400

involved in regulating growth, development, metabolism, immune function, feather molt,

401

thermoregulation, and other physiological systems. Disruption in thyroid function may have

402

implications for avian populations by contributing to reduced reproductive success. The changes

403

in thyroid function in the current study occurred in adult kestrels that were not breeding or

404

experiencing feather molt. Hence, considering the annual rhythm, physiological demands on

405

their thyroid system would have been relatively low resulting in more attenuated biological costs,

406

making contaminant-related effects on these kestrels relatively subtle. These findings are

407

consistent with the lack of apparent effects on their immune function (partially regulated by the

408

thyroid system) despite elevated hepatic EROD activity and temporarily elevated plasma

409

corticosterone levels20. Adverse effects on hematological, immune and retinol variables occurred

410

in other, similarly exposed captive American kestrels, but with longer exposure (1 month) to

411

lower levels of benzene (0.1 ppm) and toluene (0.8 ppm)21,22. In birds exposed to these types of

412

air pollutants that occur in the Athabasca oil sands region, in combination with increased

413

physiological demands from living in the wild, plus exposure to other stressors (e.g., variation in

414

food availability and quality, predation, food-borne contaminants) and environmental factors


Page 21 of 29


415

(e.g., cold temperatures, rain), the changes in thyroid function observed here in the captive

416

kestrels may occur, potentially resulting in physiological changes particularly in nestlings.

417

Further research is warranted especially in wild birds.

418 419

Acknowledgments. We appreciate the assistance of E. Cribb, P. Storino, R. Krohn, and S. Sah,

420

with the animal handling and laboratory work. We thank the staff of the Veterinary Science

421

Research Station at the University of Calgary for their excellent care and management of the

422

birds. A special thanks to France Maisonneuve (Ecotoxicology & Wildlife Health; Environment

423

and Climate Change Canada) for her analysis of the thyroid hormones. This work was

424

generously funded by a contribution agreement from Environment and Climate Change Canada

425

to the University of Calgary (KJF, JEGS), the Ecotoxicology & Wildlife Health Division (KJF),

426

a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada

427

(JEGS), and the Alberta Conservation Association (JEGS). The authors declare no competing

428

financial interest.

429



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Table of Contents Graphic.

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