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Bioaccumulation and biodistribution of selenium in metamorphosing tadpoles Chantal M. Lanctot, Tom Cresswell, Paul D. Callaghan, and Steven D. Melvin Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 19 Apr 2017 Downloaded from http://pubs.acs.org on April 21, 2017
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Environmental Science & Technology
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Bioaccumulation and biodistribution of selenium in metamorphosing tadpoles
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Chantal M. Lanctôt,*,† Tom Cresswell,‡ Paul D. Callaghan‡ and Steven D. Melvin†
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†
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4215, Australia
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‡
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Kirrawee DC, NSW 2232, Australia
Australian Rivers Institute, School of Environment, Griffith University, Southport, QLD
Australian Nuclear Science and Technology Organisation (ANSTO), Locked Bag 2001,
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* Corresponding author: Chantal Lanctôt, Australian Rivers Institute, Griffith University,
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Building G51 Edmund Rice Drive, Southport, QLD 4215, Australia. TEL. +61 07 5552 7813;
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E-mails:
[email protected],
[email protected]
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KEYWORDS
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Selenium; Selenite; Radioisotope tracer; Autoradiography; Bioaccumulation; Biodistribution;
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Amphibian; Metamorphosis
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ABSTRACT
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Selenium is an important macronutrient with a very narrow margin between
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essentiality and toxicity. Amphibians are hypothesized to be particularly sensitive due to the
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potential for metamorphosis-driven mobilization, which could transfer or concentrate
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contaminant burdens within specific organs. We explored the potential role of tissue
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degeneration and remodeling during anuran metamorphosis as a mechanism for altering
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tissue-specific Se burdens. Limnodynastes peronii tadpoles were exposed to dissolved 75Se
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(as selenite) for 7 days and depurated until completion of metamorphosis. Bioaccumulation
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and retention kinetics were assessed in whole tadpoles and excised tissues using gamma
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spectroscopy, and temporal changes in biodistribution were assessed using autoradiography.
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Tadpoles retained Se throughout metamorphosis, and partitioned the element predominantly
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within digestive and excretory tissues, including livers > mesonephros > guts > gallbladder.
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Importantly, our results demonstrate that Se biodistribution varies significantly throughout
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development. This is indicative of tissue transference, and particularly in tissues developing
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de novo after depuration. To the best of our knowledge, this is the first study demonstrating
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Se transference during metamorphic tissue remodelling. Further research is warranted to
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explore the fate and metabolism of Se (and other metal and metalloids) during anuran
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development and the implications of transference for influencing toxicity.
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TOC/ABSTRACT ART
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INTRODUCTION Selenium (Se) is a naturally occurring element that is essential for the health and
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maintenance of physiological homeostasis in vertebrates. At trace concentrations Se is a key
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component of various functional and structural proteins in the form of the selenocysteine
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(SeCys) amino acid.1 Key selenoproteins include glutathione peroxidases and thioredoxin
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reductases, are involved in cellular antioxidant defense systems and redox control, and
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iodothyronine deiodinases are involved in the activation and deactivation of thyroid
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hormones.1 However, when Se concentrations exceed what is necessary for an organism, this
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essential micronutrient can become toxic.2-4 Selenium toxicity is primarily thought to occur
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due to its similarity to sulfur (S) and tendency to erroneously substitute S during protein
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synthesis, which can lead to the distortion and dysfunction of essential biomolecules.3
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Selenium-induced toxicity may also relate to the ability for Se to generate reactive oxygen
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species resulting in oxidative stress.5
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Anthropogenic activities, including mining, agriculture and the consumption of fossil
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fuels, have been found to mobilize and release Se into surrounding waterways.6,7 Selenium
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partitioned within different compartments of aquatic ecosystems can be taken up and
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transferred in aquatic food webs from solution and diet.8,9 While dietary exposure is generally
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the dominant uptake pathway of organic Se species, dissolved Se oxyanions, selenite and
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selenate, are also readily accumulated by aquatic biota.10,11 The present study focuses on
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selenite as this oxyanion generally exhibits greater bioavailability and toxicity compared to
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selenate.11,12 Selenite is also generally more prevalent in systems receiving contaminated
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discharges from coal or oil industries, which represent a major concern for aquatic wildlife in
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Australia and globally.13 Selenium bioaccumulation in organisms inhabiting Se-laden
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systems has been linked to a range of toxic responses.3,14 The bioaccumulative properties of
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Se are therefore important for influencing risks to aquatic organisms.
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The current state of knowledge suggests that rapidly growing organisms (e.g., larval
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amphibians) and oviparous vertebrates (e.g., fish, amphibians, reptiles, birds) are the most
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sensitive to elevated Se.3,4 However, fish and birds have been the principal focus of Se
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toxicity studies,4 and much less research has considered amphibians and reptiles.3,15,16
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Amphibians have been shown to accumulate Se from contaminated water during larval
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development,11,17 but Se toxicokinetics, particularly during sensitive larval developmental
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stages, is poorly understood. This is important since amphibians may be at greater risk to
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dissolved contaminants compared to other aquatic vertebrates because of their highly
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permeable and vascularized skin, which presents an additional route of ion uptake.18
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Additionally, the lack of information on these sensitive species represents a potentially
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important shortcoming since amphibians undergo complex physiological and morphological
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changes during development, including degeneration and remodeling of virtually all larval
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tissues.19 As a consequence, it is hypothesized that accumulated trace elements may become
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mobilized and redistributed during metamorphic change, which could act as a mechanisms
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for transference amongst tissues at critical developmental stages.20 Tissue transference could
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subsequently influence the relative burden of potentially toxic trace elements, for example in
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organs prone to oxidative stress. As such, the potential for toxic elements accumulated in one
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tissue to influence concentrations in other tissues via metamorphosis-driven mobilization
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could have ramifications for influencing associated toxicity.
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We investigated uptake and retention of Se in Limnodynastes peronii tadpoles using
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radioisotope tracers, and visualized tissue-specific distributions throughout larval
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development using autoradiography. Our aim was to explore the potential role of tissue
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remodeling, degeneration and development during metamorphosis for altering tissue-specific
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contaminant burdens. Increased knowledge of these integrated processes will contribute
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greatly to our overall understanding of Se toxicokinetics in larval amphibians.
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MATERIALS AND METHODS Animals. L. peronii eggs were collected locally and tadpoles housed in 50-L aerated
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reconstituted moderately hard water (MHW).21 Laboratory conditions were maintained at
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26±1°C and 12:12h photoperiod. Tadpoles were fed Sera Micron® fry food (Sera, Heinsberg,
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Germany) ad libitum and survival monitored twice daily. Physicochemical parameters
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(temperature, pH, dissolved oxygen and electrical conductivity) were monitored before and
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after each water renewal. Methods were approved by the Animal Care and Ethics Committee
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at the Australian Nuclear Science and Technology Organisation (ANSTO), and adhered to
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the Australian Code for the Care and Use of Animals for Scientific Purposes.
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Selenium exposure and depuration. 75Se (as Na2SeO3, t1/2 = 119.8 days) was
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produced at ANSTO, Australia. Radioisotope stocks dissolved in Milli-Q water were diluted
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and equilibrated in MHW for 12 h prior to use. Forty-eight Gosner stage22 (Gs) 28 tadpoles
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were individually transferred to square 1.13 L polypropylene containers (Decor, Tellfresh)
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containing 500 mL of 75Se (23 kBq/L), equivalent to 0.6 µg Se/L (7.7 nM). Four tadpoles
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were transferred to isotope-free MHW as controls. Radiation doses were maintained
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significantly lower than LD10 for tadpoles.23,24 Tadpoles were exposed for 7 days, after which
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they were transferred to 1000 mL of Se-free MHW for 27 days of depuration. The 7-day
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uptake period was selected based on a previous study that showed that L. peronii tadpoles
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reached a steady state of Se accumulation after 7 days of exposure to SeIV.11 Aeration was
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provided to each chamber. Tadpoles were fed equal amounts of Sera Micron® twice daily
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(10% of body weight/day). Our previous study showed that feeding tadpoles in water
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containing dissolved 75Se had no effect on Se accumulation compared to tadpoles fed in clean
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water (Lanctôt et al., unpublished data), suggesting that Se did not readily bind to the food
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particles during the exposure. Exposure solutions were completely renewed daily during the
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uptake phase and for the first 3 days of depuration, and every 48 h thereafter. Waste was
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syphoned on alternate days during depuration. Subsamples of exposure solutions, collected
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before and after water renewals, were radioanalysed to verify exposure activity. Selenium
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concentration was verified using inductively coupled plasma mass spectrometry (ICP-MS;
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Varian 820MS Quadropole; run with internal standard correction for matrix and drift
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correction). Physicochemical parameters were: pH: 7.5 ± 0.2, temperature: 25°C ± 0.7, DO:
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85 ± 9.2%, conductivity: 325 ± 16 µS/cm.
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Radioanalysis. Gamma ray emissions of 75Se in water samples, tadpoles and
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standards were determined using a 1.5 × 1.5 inch LaBr detector within a lead chamber
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attached to a multichannel spectrometer (Canberra InSpector 1000). This was connected to a
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PC equipped with spectra analysis software (Genie 2000), which used varying count times
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(from 5 to 10 min) to ensure propagated counting error was 50% SeIV) in surface waters polluted by coal and oil
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industries.13,30 A low concentration was targeted as we wished to evaluate Se
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bioconcentration in the absence of toxicological responses and limited information is
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available on sub-lethal impacts of dissolved Se on amphibian larvae.
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Survival, development and condition. Se (as selenite) had no influence on tadpole
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survival, with only a single mortality occurring throughout the experiment. Control tadpoles
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reached metamorphic climax (Gs 42) slightly faster (~ 3d) than treated tadpoles (U = 2.07, p
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= 0.041), but tadpoles from both groups completed metamorphosis (Gs 46) within a similar
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timeframe (U = 1.84, p = 0.076; data not shown). Similarly, condition (K) did not differ
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between groups at the end of the experiment (t = 1.10, df = 8, p = 0.3318, data not shown).
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The assessment of K throughout development revealed a significant decrease during
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metamorphic climax (Gs 42 to Gs 46; Figure S1; p < 0.0001). This is not surprising as
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tadpoles cease feeding at the onset of metamorphic climax when their digestive tract and
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craniofacial structures are restructured for terrestrial life. Reduced mass during this energy
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intensive phase could influence Se burdens by concentrating Se within specific tissues. This
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hypothesis is supported by Snodgrass and collaborators who reported increased Se body
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burdens in Rana catesbeiana and R. clamitans metamorphs relative to larval stages following
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exposures to coal combustion waste.20,31
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Tadpole uptake and retention kinetics. Following 7 days exposure, tadpoles
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contained 0.27 ± 0.08 µg Se/g wet weight (ww) (3.4 ± 1.0 nmol Se/g ww; mean ± SD; Figure
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1) and BCF was 425 ± 125 mL/g ww (mean ± SD; Table 1). Whole body Se content is
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equivalent to approximately 1.9 µg Se/g dry weight (dw) based on 86% moisture content
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calculated from control animals (data not shown). After being transferred to clean water, Se
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was significantly depurated over time (Figure 1; F(5,33) = 32.33, p < 0.0001), with whole-body
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depuration kinetics best described by a one-phase exponential decay model (Figure 1 and
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Table 1). Specifically, 42% of accumulated Se was depurated in the first 3 days and a further
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41% was depurated over the following 10 days (total loss of 83%) (p < 0.05). After 10 days
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of depuration, Se reached a steady-state, with only 10 to 14% of the initial Se remaining to
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the end of the experiment (p > 0.05). On average (± SD) 0.03 ± 0.02 µg Se/g ww (0.38 ± 0.25
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nmol Se/g ww) was retained by tadpoles after depuration.
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Results are consistent with one earlier study assessing uptake and depuration kinetics
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of 75Se in Xenopus laevis embryos and hatchlings, where embryos were exposed to 5000 µg
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Se/L (as selenite and selenous acid) for up to 48 h.17 In that study, rapid linear uptake of Se
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was followed by a rapid initial loss of approximately 80% of Se burdens within 30 mins of
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depuration, and animals reached a steady-state after 3 d with 10 to 15% of Se being retained.
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Despite a potential for Se accumulation to be inflated by Se bound to the outer jelly layer of
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embryos, depuration kinetics and retention in Xenopus embryos and hatchlings showed
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similar trends as tadpoles in our study (exposed between Gs 28 to Gs 31). Contrary to this,
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our previous work showed that Gs 25 tadpoles (L. peronii) exposed to selenite for 4 days
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retained greater than 80% of Se following depuration for up to 72 h, and tadpoles appeared to
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have reached a steady state during this short depuration time (Lanctôt et al., unpublished
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data). This suggests that early post-embryonic stages may be more susceptible to Se uptake
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compared to embryonic and later larval stages. Differences in sensitivities amongst
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amphibian stages have been demonstrated by several studies.17,32,33 These studies are
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generally in agreement that early larval stages are more sensitive than embryos or later
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stages.
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Our results show that tadpoles retain Se burdens throughout metamorphosis, which
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likely stems from its ability to replace S in biological molecules.20 This corroborates previous
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field studies, which found that Se was retained in metamorphs when larvae were exposed to
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coal-contamination, unlike most other trace elements.20,31,34 Similarly, another study reported
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no loss of Se between larvae and metamorphs when animals were fed Se (as SeO2) as
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tadpoles.35 As such, Se burdens in larvae may also contribute to toxicity in juvenile and adult
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frogs, which are expected to continue accumulating Se through their diet.16,34 This holds
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significant implications for amphibian populations, since Se exposures have been linked to
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severe impacts on survival, behaviour, reproduction and deformities.15-17,31,36-39
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Tissue-specific biokinetics and distribution throughout metamorphosis. Given the
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drastic changes that almost every organ and tissue undergoes during metamorphosis, it is
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crucial to understand the potential impacts of these changes on tissue burdens. Not
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surprisingly, Se bioconcentration was found to differ significantly among dissected tissues
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(Table 1; F(9,28) = 15.0, p < 0.0001), but depuration kinetics followed a similar trend to whole
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tadpoles (R2 = 0.81 ± 0.08; Table 1 and Figure S2). Though most organs exhibited similar
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depuration kinetics, relative proportions of Se burdens amongst some organs varied
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significantly throughout development (Figure 2). Temporal changes observed in Se
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biodistribution are hypothesized to reflect tissue transference resulting from the three
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transformative processes that occur during metamorphosis: remodeling, resorption and de
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novo tissue development.40 Descriptions of selenium accumulation and metamorphic changes
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exhibited in each of the major sites of accumulation are described below.
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Digestive and excretory organs. At the end of the uptake phase and throughout
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depuration, Se was predominantly located in digestive and excretory tissues, including liver >
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mesonephros > gut > gallbladder, which had the highest BCF values relative to other tissues
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(Table 1). The predominant partitioning of Se in these organs was confirmed via
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autoradiography (Figure 3), and is consistent with previous fish studies.41-43 Accumulation in
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these organs likely reflects key elimination routes in tadpoles, which include excretion via the
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liver, intestine, mesonephros and gallbladder, and shedding of apoptotic cells.20
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The liver of tadpoles, like all vertebrates, is involved in absorption, processing and
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storage of nutrients from the digestive tract, and is the site of important metabolic processes
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like protein synthesis, bile secretion, urea synthesis and detoxification. Predominant Se
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accumulation in the liver is thus not surprising since this organ serves to process this element
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for protein incorporation, detoxification or elimination.44 Importantly, studies have shown
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that hepatic Se can bind to the sulfur-containing protein vitellogenin (VTG) in the liver of
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oviparous animals.3 Selenium containing VTG is consequently maternally transferred to
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embryos through vitellogenesis and yolk resorption, causing a range of reproductive
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defects.3,45 However, little is known on Se metabolism in the liver of tadpoles.
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Biodistribution results revealed that the relative proportion of Se in the liver (Figure
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2a; F(5,18) = 9.69, p < 0.0001) and gallbladder (Figure 2i; H = 16.49, df = 5, p = 0.0056)
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increased by 3- to 4-fold throughout development. Despite BCF being lower in gallbladder,
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gallbladders had the greatest retention efficiencies and retained 2 to 4 times more Se
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compared to liver, gut and mesonephros (Table 1). Selenium elimination through bile
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excretion has also been demonstration in other species.46
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Contrary to the increasing Se distribution in the liver and gallbladder, the gut and
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mesonephros experienced the most drastic reductions in Se concentrations throughout
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metamorphosis, with final retentions of only 6 and 7%, respectively (Table 1). The
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proportion of Se in the gut was significantly reduced by 8- to 22-fold between Gs 31 and
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metamorphic climax (Figure 2c; F(5,18) = 162.68, p < 0.0001). The reduction in relative gut
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Se, especially in later stages, reflects drastic gut remodeling occurring during
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metamorphosis.47 In fact, during the final stages of development (Gs 41 to Gs 46), primary
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(larval) epithelial cells are completely removed through apoptosis and replaced by secondary
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(adult) epithelial cells that form a more complex multi-folded epithelium.48,49 During this
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time, tadpoles cease feeding and the gut is significantly reduced in size (approximately 75%
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reduction in length).19 While the decrease in relative Se abundance in the gut of metamorphic
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stages can be explained by dramatic tissue remodeling, the decrease observed during the
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growth phase of earlier stages (Gs 31 to Gs 37) indicates that Se accumulated in the gut was
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either eliminated or absorbed. The role of the tadpole gut in sequestering and incorporating
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Se into other tissues warrants further investigation.
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The tadpole mesonephros (also called opisthonephric kidneys) primarily serves to
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eliminate waste and regulate water, ions and acid-base balance. The mesonephros of tadpoles
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undergoes significant change to shift from ammonia to urea production during
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metamorphosis. In fish, kidneys are a primary site for Se accumulation and urine the primary
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route of excretion.43,46 Though Se bioconcentration in tadpole mesonephros was quite high
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relative to other tissues at the end of the uptake phase, retention was relatively low
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(approximately 7%). This suggests that tadpoles excrete Se via the mesonephros. Moreover,
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biodistribution results show a two-fold increase in the mesonephros in early stages (between
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Gs 31 and Gs 33; F(5,18) = 9.33, p < 0.0001) but no significant differences were observed
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between Gs 31 and later stages (Gs 37 to Gs 46) (Figure 2b). Despite their role in Se
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excretion, accumulation of excess Se can have damaging consequences on renal and hepatic
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tissues, including necrosis, increased hepatosomatic index, increased number of Kupffer cells
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in the liver and mesangial cells in kidneys, and decreased hepatic reduced glutathione.50-52
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Peripheral tissues. Whole tails, which include the epidermis, muscle, connective
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tissue, spinal cord and notochord, accumulated the lowest concentration of Se at the end of
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the uptake phase compared to other tissues (Table 1 and Figure S2g). Autoradiographs
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revealed Se to be predominantly accumulated within the somitic striated tail muscles (Figure
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3). Despite relatively low Se burdens the tails retained 2 to 4 times more Se than other tissues
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through depuration (Table 1). Additionally, the relative proportion of Se in tails increased as
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tadpoles developed from Gs 31 to Gs 42, but decreased post-metamorphic climax (Figure 2g;
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F(4,18) = 27.40, p < 0.0001). Reduction of Se in larval-specific organs such as tails (and gills)
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during metamorphosis is expected since these tissues degenerate at the onset of metamorphic
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climax.53 Importantly, Se associated with apoptotic cells may be either eliminated and/or
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redistributed during this process. Transference of Se burdens from degenerated cells to
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surrounding cells may lead to concentrated tissue burdens. This could be linked to previously
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observed tissue damages reported in X. laevis tadpoles exposed to dissolved Se (2 to 10 mg
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Se/L as selenite), which showed morphological anomalies in the somites, including the
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disorganization and degeneration of myonemes and myofibrils and mitochondrial swelling.54
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Contrary to tadpole tails that start degenerating at metamorphic climax, the limbs,
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including both muscle and cartilage/bone, develop continuously throughout development.
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Biodistribution results reflect this difference, showing a linear increase of Se in the hind
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limbs until completion of metamorphosis (Figure 2f; F(5,18) = 630.25, p < 0.0001).
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Additionally, autoradiographs reveal Se incorporation to be relatively homogeneous amongst
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skeleton and muscle (Figure 3). Though accumulation was relatively low in tadpole limbs,
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incorporation of Se in the developing limbs during depuration is clear evidence of tissue
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transference. The sequestering of Se in peripheral tissues may be linked to plasma and other
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selenoproteins in the form of SeCys, including selenoprotein P, N and W, and iodothyronine
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deiodinase enzymes, associated with skeletal muscle.55,56 Further research is warranted to
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investigate the role of these proteins in Se transference during metamorphosis. Of particular
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interest are deiodinase enzymes that are essential for the regulation of local thyroid hormone
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(TH) activity during amphibian metamorphosis.57,58 TH plays a central role in tadpole
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development by triggering proliferation of adult progenitor/stem cells and degeneration and
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remodeling of larva-specific tissues and organs.59 As such, larval amphibians may be
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expected to be particularly sensitive to deficiencies or excess of Se in the environment, due to
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the important role of deiodinases in the coordination of thyroid-mediated metamorphosis.58 In
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humans, excess Se has been linked to decrease serum T3 levels,60,61 which may relate to
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disruption of deiodinase enzymes.62-64 Future research is necessary to determine the impacts
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of Se on deiodinase expression and function in developing amphibians as this may help
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explain previous reports of Se toxicity in developing embryos and tadpoles.17,37,39,54
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Sensory organs. The relative proportion of Se within tadpole eyes increased linearly
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throughout development (Figure 2d; H = 18.12, df = 5, p = 0.0028). Autoradiographs reveal
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that Se was accumulated predominantly within the ocular lens, with lower levels in the retina
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(Figure 3). To our knowledge, this is the first report of selenium accumulation in amphibian
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eyes. A recent study similarly demonstrated Se accumulation within the lens-core and lens-
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epithelium of zebrafish (Danio rerio) larvae exposed in ovo through maternal transfer of
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dietary SeMet (30 µg Se/g dw).65 Importantly, differences in exposure route (waterborne vs.
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in ovo) and Se species (selenite vs. SeMet) did not influence Se partitioning within the eye
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lens in larval amphibians and fish. Confocal x-ray absorption spectroscopy showed Se
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accumulation in the form of a selenomethionine-like species in zebrafish.65 Despite tadpoles
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being exposed to a different form in the present study, it is likely that Se was also
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incorporated in the tadpole lens in the form of selenomethionine. Nonetheless, speciation
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assessments are necessary to confirm the fate of Se accumulated in the tadpole eye and
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explore changes that might occur through metamorphosis.
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Selenium accumulation observed in the ocular lens in this and other studies may
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explain Se-induced ocular impairment reported in several species. 15,66-69 In particular, a
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recent study describing maternal transfer of dietary SeMet in X. laevis showed that lens
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abnormalities were the most sensitive indicator of in ovo exposure (EC10 = 43.4 µg Se/g egg
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dw).15 Ocular lens malformation reported in that study included a reduction in spherical
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shape, cataract-like opaqueness and ectopy from the ocular globe.15 Severe craniofacial
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abnormalities related to the eyes were also documented at the highest exposure concentration
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(90 µg Se/g dw in maternal diet).15 Several different mechanisms for Se-induced ocular
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impairments have been proposed, including the potential interference of Se with Pax6
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transcription factors,15 formation of oxidized gluthathione,66,68 accelerated apoptosis, 70,71 and
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incorporation of Se into lens proteins.65,68,72. The most recent evidence, however, points
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towards the latter. Choudhury and colleagues hypothesized that ocular impairments are
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caused by increased oxidation resulting from selenomethionine incorporation into crystalline
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proteins.65 The hypothesis is derived from the fact that selenomethionine is more readily
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oxidized than methionine and that oxidation is known to contribute to cataract formation.65,73
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These hypothesized mechanisms may also apply to amphibians, but more research is needed
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to elucidate the mechanisms of ocular deformities in these species.
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Contrary to the eyes, the ears did not appear to be major site of Se accumulation
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(Figure 3). This confirms confocal x-ray fluorescence images of Se in zebrafish larvae, which
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showed no major Se accumulation in the ears.65 Other sensory systems were not assessed in
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this study.
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Respiratory organs. Tadpoles use gills, lungs and skin for respiration, and can
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exchange gases with both air and water. The high permeability of the respiratory organs in
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larval amphibians poses a concern for contaminant uptake. In normoxic conditions, gills and
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skin contribute to the majority of gas exchanges in larval stages (< Gs 42).74 The lungs, on
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the other hand, dominate oxygen intake at later stages (> Gs 45), although skin remains the
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primary site of CO2 elimination.74 Changes in environmental conditions, such as hypoxia and
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temperature, have been shown to alter the relative roles of these organs in gas exchange.75 In
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the present study, dissolved oxygen was relatively stable (85 ± 9.2%), and thus gas exchange
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was presumed to occur as under normoxic conditions.
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By the start of the exposure, tadpoles (Gs 28) had regressed their outer transient gills,
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and possessed only internal gills. Autoradiographs reveal Se accumulation predominantly
381
within the gill tufts, which consist of epidermis, connective tissue, endodermal-pharyngeal
382
cells, blood vessels and capillaries or associated branchial cartilage and muscles (Figure
383
3a).76 Apoptosis of the internal gills is initiated at the onset of metamorphic climax and
384
occurs through completion of metamorphosis.53 This is reflected by a decrease in the relative
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proportion of Se in the gills during late development (Figure 2h; H = 13.46, df = 4, p =
386
0.0092). Though gills do not represent a site of long-term storage, Se at higher concentrations
387
can cause damage to gill structures and affect respiration. For example, Se pollution has been
388
linked to gill malformations in fish, including swollen lamellae and haemorrhages, that can
389
impair their respiratory capacity and lead to metabolic stress.50
390
Tadpole skin undergoes considerable changes during development, with increasing
391
number of epidermal cell layers and increasing vascularization as larvae progress through
392
metamorphosis.77 Similar to the epidermal changes that occur to the gut, larval skin cells are
393
completely replaced by adult cells during metamorphosis. The resolution of autoradiographs
394
did not permit a thorough assessment of Se uptake within or through the epidermis, but the
395
skin does not appear to be a major site of Se accumulation in tadpoles. In the present study,
396
Se concentrations in the skin were quantified only at the end of the experiment. We found
397
that metamorphs retained 0.022 ± 0.01 µg/g ww of Se, which is comparable to the amount of
398
Se measured in the brain, limbs, lungs and gut at this stage. Nonetheless, cytotoxic effects,
399
including the disorganization, vacuolization and swelling of the outer layer of epithelial cells
400
and mitochondria, have been reported in X. laevis embryos and early post-embryonic larvae
401
exposed to dissolved Se (as selenite) at higher concentrations (2 to 10 mg/L).54
402
Similar to the skin, lung structure and vascularization progresses throughout
403
development. Selenium accumulation in the lungs was relatively low, and Se concentrations
404
at the end of the depuration phase were similar to that observed in the skin (Figure S2).
405
Contrary to gills and skin, the proportion of Se in the lungs peaked at Gs 42 (Figure 2j; H =
406
13.40, df = 5, p = 0.0199). This could reflect the increasing role of lungs in oxygen
407
consumption as gills start to regress. However, this measurement was highly variable and
408
post-hoc analysis found no differences amongst stages (p > 0.05).
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Central nervous system. The tadpole brain consists of three main regions: the
410
forebrain (telencephalon and diencephalon), midbrain (mesencephalon) and hindbrain
411
(rhombencephalon), which can be distinguished in the optical images shown in Figure 3.
412
Whole brain analysis showed relatively low Se accumulation compared to other organs
413
(Table 1 and Figure S2) and revealed that the relative proportion of Se in the brain was not
414
significantly altered throughout development (Figure 2e; F(5,18) = 1.06, p = 0.415).
415
Autoradiographs confirm that Se burdens in the brain are relatively low (Figure 3), though
416
images reveal that Se is not homogeneously accumulated within the CNS. Selenium appears
417
to be partitioned predominantly to the forebrain and the spinal cord (Figure 3).
418
Circulatory system. Absorbed Se is generally transported to target tissues via the
419
circulatory system by plasma proteins.41,78 Though circulating concentrations were not
420
assessed in the present study, an earlier study found relatively low selenite concentrations in
421
fish blood compared to selenate, suggesting that the former has a greater tissue affinity.41 In
422
agreement with these findings, autoradiograph images revealed that the heart, including blood
423
and cardiac muscle, was not a major site of Se accumulation at low exposure concentrations.
424
Nonetheless, circulating and cardiac accumulation should be considered in future studies as
425
excess Se was found to induce cardiac defects, anemia, reduced hematocrit, hemoglobin and
426
erythrocites in other vertebrates.50,79
427
Implications. To the best of our knowledge, ours is the first study to demonstrate Se
428
transference during metamorphic tissue remodelling. Considering the relatively narrow
429
window between essentiality and toxicity, tissue transference may be an important factor that
430
could influence whether Se burden surpasses toxic thresholds. As such, the mobilisation and
431
transference of contaminant burdens during metamorphosis may provide an explanation for
432
the increased sensitivity of larval amphibians during this critical life stage. Further research is
433
necessary to explore this phenomenon with exposure to elevated Se concentrations, such as
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that representative of Se-laden industrial sites, as well as with dietary exposure of different Se
435
species, and to identify whether tissue transference occurs with other pollutants. Our findings
436
support other work showing that Se partitions within the eye lens of larval amphibians and
437
fish, regardless of exposure route and Se species. The significance of Se within the eye lens
438
of aquatic organisms requires more research as this could affect their ability to reproduce,
439
detect preys and avoid predators, which could have serious implications at a population level.
440
Knowledge of biodistribution is essential to understand risks associated with exposure, and
441
more research is therefore needed to explore tissue-specific fate and metabolism of important
442
pollutants in sensitive species undergoing metamorphosis.
443 444
ACKNOWLEDGEMENTS
445
The study was carried out at ANSTO with the assistance of an Australian Institute for
446
Nuclear Science and Engineering Research Award (No. ALNGRA16029). We thank An
447
Nguyen and Zoe Williams for sectioning and autoradiography, Lida Mokhber-Shahin for
448
analysis of standards, Henri Wong and Brett Rowling for ICP-MS analysis, and Nicholas
449
Howell for tissue radioanalysis.
450 451
SUPPORTING INFORMATION
452
Supporting information is available free of
charge at http://pubs.acs.org. Detailed methods
453
of tissue digestion and autoradiograph standard preparation and analysis. Additional figures
454
of condition factor (Figure S1) and depuration kinetics and retention Se in tissues (Figure
455
S2).
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Commun. 2000, 275 (2), 300–306. Belusko, P. B.; Nakajima, T.; Azuma, M.; Shearer, T. R. Expression changes in mRNAs and mitochondrial damage in lens epithelial cells with selenite. Biochim. Biophys. Acta 2003, 1623 (2-3), 135–142. Palsamy, P.; Bidasee, K. R.; Shinohara, T. Selenite cataracts: Activation of endoplasmic reticulum stress and loss of Nrf2/Keap1-dependent stress protection. Biochim. Biophys. Acta 2014, 1842 (9), 1794–1805. Kantorow, M.; Hawse, J. R.; Cowell, T. L.; Benhamed, S.; Pizarro, G. O.; Reddy, V. N.; Hejtmancik, J. F. Methionine sulfoxide reductase A is important for lens cell viability and resistance to oxidative stress. PNAS 2004, 101 (26), 9654–9659. Burggren, W. W.; West, N. H. Changing respiratory importance of gills, lungs and skin during metamorphosis in the bullfrog Rana catesbeiana. Respir. Physiol. 1982, 47, 151–164. Crowder, W. C.; Nie, M.; Ultsch, G. R. Oxygen uptake in bullfrog tadpoles (Rana catesbeiana). J. Exp. Zool. 1998, 280, 121–134. Brunelli, E.; Perrotta, E.; Tripepi, S. Ultrastructure and development of the gills in Rana dalmatina (Amphibia, Anura). Zoomorphology 2004, 123, 203–211. Schreiber, A. M.; Brown, D. D. Tadpole skin dies autonomously in response to thyroid hormone at metamorphosis. PNAS 2003, 100 (4), 1769–1774. Sandholm, M. Function of erythrocytes in attaching selenite-Se onto specific plasma proteins. Acta Pharmacol. Toxicol. 1975, 36, 321–327. Ma, Y.; Wu, M.; Li, D.; Li, X.-Q.; Li, P.; Zhao, J.; Luo, M.-N.; Guo, C.-L.; Gao, X.B.; Lu, C.-L.; et al. Embryonic developmental toxicity of selenite in zebrafish (Danio rerio) and prevention with folic acid. Food Chem. Toxicol. 2012, 50, 2854–2863.
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Table 1. Bioconcentration factor (BCF, mL/g wet weight), retention efficiency (RE, %),
681
elimination rate (ke, d –1) and depuration curve half time (t1/2, d) of Se in whole tadpoles
682
exposed for 7 days followed by 27 days depuration in clean water. Values are mean ± SE.
683
Letters indicate significant differences in BCF among tissues, based on Tukey HSD post hoc
684
test with α = 0.05. BCF Whole body 425 ± 62 Liver 1950 ± 283a Mesonephros 1435 ± 239ab Gut 878 ± 190bc Gallbladder 549 ± 24cd Eyes 520 ± 79cd Gills 452 ± 74cd Legs 422 ± 111cd Lungs 375 ± 83cd Brain 252 ± 26cd Tail 134 ± 19d
ke 0.22 ± 0.04 0.30 ± 0.08 0.17 ± 0.04 0.57 ± 0.18 0.25 ± 0.12 0.18 ± 0.05 0.30 ± 0.08 0.35 ± 0.12 0.16 ± 0.07 0.21 ± 0.04 0.36 ± 0.14
RE 10.3 ± 3.3 11.1 ± 4.1 6.8 ± 5.3 6.3 ± 4.5 23.0 ± 6.4 8.9 ± 5.0 8.6 ± 5.0 6.6 ± 5.5 14.1 ± 8.7 12.1 ± 3.3 23.4 ± 5.8
t1/2 3.09 2.33 4.08 1.23 2.77 3.84 2.29 2.00 4.27 3.30 1.95
R2 0.83 0.85 0.88 0.83 0.66 0.87 0.86 0.76 0.72 0.92 0.75
685 Gosner stage 0.4
31 33
37
42 44
46
a
150 125
µg Se/g ww
100
b
0.2
75 50
0.1
c
c
c
c 25
0.0
0 0
686
% Retention
0.3
3
6
9
12 15 18 21 24 27
Depuration time (d)
687
Figure 1. Concentration (µg Se/g wet weight; left) and retention (%; right) of Se in whole
688
tadpoles exposed for 7 days and sampled at different developmental stages (Gosner, 1960)
689
throughout 27 days of depuration in clean water. Values are means ± SE (n = 4-7). Line
690
shows the best-fit one-phase exponential decay (R2 = 0.83). Letters indicate significant
691
differences among developmental stages, based on Tukey HSD post hoc test with α = 0.05.
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Biodistribution (%)
40
b) Mesonephros
Gosner stage
31 33
37
42 44
30
c
20
bc
ab
bc
10
46
bc
10 a
Biodistribution (%)
a) Liver
0
Gosner stage
31 33
8
37
3
6
9
bc ab
4 ab
a
0
3
6
Biodistribution (%)
d) Eyes
Gosner stage 31 33
37
42 44
b c 20
de d
8
46
e
0
Gosner stage
31 33
37
3
6
e) Brain
9
ab
ab
ab
ab 2
a
0
3
6
f) Hind limbs
Gosner stage
31 33
37
9
12 15 18 21 24 27
Depuration time (d)
42 44
30
46
Biodistribution (%)
Biodistribution (%)
46
b
4
12 15 18 21 24 27
2
1
Gosner stage
31 33
37
42 44
46
d d
20
c 10
a
a
0
3
b
0
0 0
3
6
9
12 15 18 21 24 27
6
g) Tail
h) Gills
Gosner stage 37
42 44
b
30 20
a
b
a
10
46
Biodistribution (%)
31 33
9
12 15 18 21 24 27
Depuration time (d)
Depuration time (d)
Biodistribution (%)
42 44
6
Depuration time (d)
a
10 0
Gosner stage 31 33
8 ab
37
42 44
ab
ab
46
a
6 4 2
b
0 0
3
6
9
12 15 18 21 24 27
0
3
6
Depuration time (d) j) Lungs
Gosner stage
31 33
37
42 44
b
2
9
12 15 18 21 24 27
Depuration time (d)
b
ab 1 ab ab
a 0
Gosner stage
5
46
31 33
Biodistribution (%)
i) Gallbladder
Biodistribution (%)
12 15 18 21 24 27
0
0
37
42 44
46
4 3 2 1 0
0
692
9
Depuration time (d)
40 a
3
ab
2
12 15 18 21 24 27
Biodistribution (%)
c) Gut
40
46
c
6
Depuration time (d)
3
42 44
0 0
60
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3
6
9
12 15 18 21 24 27
Depuration time (d)
0
3
6
9
12 15 18 21 24 27
Depuration time (d)
693
Figure 2. Proportion (%) of Se in tissues (µg Se/organ) relative to total body concentrations
694
(i.e., sum of all tissues) throughout metamorphosis. Boxplots show interquartile range
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695
(column), median (horizontal line), minimum and maximum values (whiskers) and average
696
(+) of 3-4 replicates. Letters indicate significant differences among developmental stages (α =
697
0.05)
698
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699
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700
Figure 3. Digital autoradiographic phosphor image and optical image of block face (right and
701
left, respectively, in each panel) showing Se distribution in ventral (left) and dorsal (right)
702
sections of tadpole at Gosner stage 33 (a-b), Gs 42 (c-d), Gs 44 (e-f) and 46 (g-h), sampled
703
after 3, 19, 22 and 27 of depuration in clean water, respectively. B, bone; BR, brain; EL, eye
704
lens; FB, fat bodies; FL, forelimb muscle; G, gallbladder; GI, internal gills; GU, gut; H,
705
heart; HL, hind limb muscle; IE, inner ear; L, liver; LU, lung; M, mesonephros; MO, mouth;
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MU, muscle; N, notochord; R, retina; S, stomach; SC, spinal chord; TM, tail muscle.
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