Aop: 473

Title

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Energy deposition from internalized Ra-226 decay leads to altered ventilation behavior and slower growth in Lymnaea stagnalis due to apparent hypoxia from decreased hemocyanin oxygen binding capacity

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Energy deposition from Ra226 decay lowers oxygen binding capacity of hemocyanin

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Authors

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Danielle Beaton, Canadian Nuclear Laboratories

Point of Contact

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Danielle Beaton   (email point of contact)

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  • Danielle Beaton

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Author status OECD status OECD project SAAOP status
Under development: Not open for comment. Do not cite
This AOP was last modified on November 21, 2022 16:37

Revision dates for related pages

Page Revision Date/Time
Deposition of Energy October 17, 2022 16:40
Increase in reactive oxygen and nitrogen species (RONS) May 08, 2019 12:30
increase oxidation of the di-copper moiety of the hemocyanin active site November 16, 2022 11:37
Decreased, oxygen binding capacity by hemocyanin November 16, 2022 12:10
Cognitive, sensed as hypoxic/low oxygen environment November 16, 2022 12:28
Increase, hemocyanin mRNA November 16, 2022 12:32
Increase, pulmonate breathing November 16, 2022 12:38
Decrease, Growth July 06, 2022 07:36
Decreased, Reproductive Success December 03, 2016 16:37
Energy Deposition leads to Increase in RONS March 28, 2022 07:19
Increase in RONS leads to methemocyanin formation (decrease overall oxygen binding capacity) November 16, 2022 12:49
methemocyanin formation (decrease overall oxygen binding capacity) leads to Decrease overall oxygen binding capacity (methemocyanin formation) November 16, 2022 12:50
methemocyanin formation (decrease overall oxygen binding capacity) leads to Hemocyanin Bohr effect decrease November 16, 2022 12:51
Hemocyanin Bohr effect decrease leads to behavioral change leading to possible reduced feeding opportunity November 16, 2022 12:52
behavioral change leading to possible reduced feeding opportunity leads to Decrease, Growth November 16, 2022 12:53
Decrease overall oxygen binding capacity (methemocyanin formation) leads to Increase, hemocyanin mRNA November 16, 2022 12:54
Decrease, Growth leads to Decreased, Reproductive Success November 16, 2022 12:59

Abstract

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This AOP features the freshwater giant pond snail, Lymnaea stagnalis, as an example freshwater aquatic organism and one that is included as a test organism for setting water quality criteria.  The Giant pond snail is an intermediary organism in freshwater ecosystems where it consumes vegetation, algae and decaying organic matter in the lower trophic levels and is a source of food to fish in the higher trophic levels. They are geographically widespread, air breathing (pulmonate), simultaneous hermaphroditic gastropods with simple nervous systems and well-characterized life cycles [2]. This makes them useful research species in understanding facets of the neurobiology of learning and motivation and in toxicology research. Male maturation takes ~30 days and female maturation takes ~60 days.  In the wild their lifespan is roughly one year [3] [4]. The Lymnaea shell forms from the mantle where an organic matrix is secreted, forming a scaffold for calcium carbonate biomineralization akin to that found for the oyster [5] [6]. 

Calcium and other alklaine earth elements from the environment are taken up by the snail. Throughout its soft parts, calcium stores help to maintain a constant calcium concentration within the the snail's hemolymph.  Uptake of the alkaline earth radionuclide radium-226 from the environment follows the dynamics of calcium distribution and storage, suggesting that the hemolymph may offer a way to test for possible early events that follow from the decay of internalized radium-226 related to growth, reproduction and survival.  This AOP proposes that decay of internalized radium-226 produces radiolysis products that react with the snail's oxygen carrying protein, hemocyanin, with the effect of lowering its oxygen binding capacity.  This change is interpreted as hypoxia motivating the snail to engage in longer and/or more frequent pulmonate breathing.  This change, in turn, could lower feeding opportunity and thus lower growth.

AOP Development Strategy

Context

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Changes in Hemocyanin as an Early Event following Energy Deposition

The Giant pond snail uses the copper bearing protein, hemocyanin, for oxygen uptake and delivery to cells. The copper elements at the oxygen binding center of this protein display a characteristic UV-visible spectrum depending on whether the copper moiety is bound to oxygen or not.  In transitioning from its deoxygenated form to its oxygenated form, the hemocyanin protein changes from colorless to blue with an UV-visible absorption peak at ~348 nm [29]. For this reason, species with hemocyanin are referred to as “blue blooded”.  

In its met‑hemocyanin form, the oxidized copper moiety cannot bind oxygen as measured by the disapearance of tthe UV-visible absorption peak at 348 nm and the appearance of a low broad peak around 380-410 nm [29].  Observable changes in the hemocyanin UV-visible strectrum upon the oxidation of copper suggest it may be possibleto connect this change in hemocyanin properties to the oxidizing nature of radiolysis products formed following energy depositon (onto biological matter) from ionizing radiation.  

Calcium uptake as a proxy for Ra-226 uptake:  Justification to Study Proteins in Hemolymph

The snail acquires calcium from the environment where uptake follows saturable Michaelis Menten type kinetics with a half saturation coefficient of 0.3 mM in artificial tap water [7]. After uptake, the calcium ion enters the hemolymph, where it is taken up by other organs and used in cellular ATP production. In this study on L. stagnalis physiology, it was found that the calcium ion activity in hemolymph was lower than what would be predicted by the Debye-Huckel theory [8], suggesting that the bicarbonate ion produced as part of cellular metabolism forms complexes with some of the internalized calcium ions. In addition to the hard parts of the body, calcium carbonate deposits distribute through the soft parts of the snail's body within ‘calcium cells’ (aka rhogocytes, pore cells) that line the blood vessels, connective tissue, the foot muscle and the digestive gland [9].

Uptake of Ra-226 would distribute thoughout the snail body in a similar manner to the distribution of calcium, suggesting that a proportion of the internalized Ra‑226 resides within the soft tissues where calcium cells/rhogocytes are located. This accumulation is akin to Ra-226 uptake and distribution into granules within a freshwater muscle [10].

An electrochemical potential difference between calcium in the medium and calcium in the hemolymph varies with the external calcium [7]. The corresponding equilibrium potential suggests that there is a positive calcium balance within the snail hemolymph when the calcium concentration in the medium is 0.062 mM or higher. Uptake is likely an active process operating against an electrochemical gradient at external calcium concentrations of 0.062-0.5 mM, while uptake occurs with minimal free energy change in the absence of an electrochemical gradient at external calcium concentrations higher than 0.5 mM.

The accumulation half-life for calcim in hemolymph is around 20 to 24 hours, eventually achieving ~70% of calcium content of the medium [11] . Lymnaea stagnalis grown in a medium of both calcium and Ra-226 also accumulated ~70% of the Ra-226 activity [12], further suggesting that the distribution and dynamics of Ra-226 within the snail body corresponds to the distribution and dynamics of calcium. Studies using labeled calcium found that the rate of calcium accumulation into the shell was slower than its accumulation into the blood, but that eventually the shell, as the main location in the snail for biomineralization, accumulated most of the labeled calcium.

Notably, the dynamics of calcium uptake, storage and loss work to maintain a constant blood calcium concentration and calcium flux [11], even under conditions of variable calcium in the medium [13]. This “calcium buffer” system is linked to how bicarbonate is handled in the hemolymph such that when calcium in the environment is present in excess, some of the calcium is stored as calcium carbonate within the calcium cells/rhogocytes and the shell . However, at times when calcium from the environment is lacking, stored calcium from these reserves is returned to the hemolymph, thus maintaining a stable circulating calcium concentration.

Possible Early Biological Events from Internalized Radium-226 in the Snail, Lymnaea Stagnalis

The MIE for ionizing radiation is energy deposition.  This event is followed by tracks of ionization and excitation events as the ionizing particles traverse the biological medium, which is largely composed of water.

At the sites of ionizations and excitations along the particle track, the ensuing dissipation of energy via the abundant secondary electrons contributes to the radiolytic formation of hydrated electrons, hydroxyl radicals, hydride radicals and superoxide radicals. These in turn further react to form the final yields of reactive and molecule species, including hydrogen gas and hydrogen peroxide [14]. The combination of direct physical events and indirect physicochemical events set the stage for biological events and possible radiation-induced adverse outcomes.

The snails's soft tissues are bathed in hemolymph [REF]. A major protein in hemolymph is hemocyanin.

Rhogocytes are the cells that express hemocyanin [REF] [REF] and they are the sites of soft tissue calcium storage [REF]. Increased hemocyanin RNA expression is observed via RNA in situ hybridization of rhogocyte histology slides ##########

Histology and bulk tissue anlysis of freshwater mussels show that Ra-226 accumulates within calcium granules in mussel soft tissues to values greater than the external Ra-226 [Jefferee and Simpson, 1984, ######], [Brenner, Smoak et al 2007, Limnol Oceanogr 52, 1614-1623].  Alpha track imaging of Ra-226 decay overlaying histological images of the mussel localized alpha particle tracks to the calcium granules (Ra-226) and nearby soft tissues possibly from additional alpha tracks emitted from decay of the Ra-226 daughters:  the mobile Rn-222 gas and Po-218, Po-214 and Po-210 or other alpha emitting radionuclides.

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A shift from oxy/deoxy hemocyanin to methemocyanin would be a sign that the exposed snail's physiology has changed and thus may be a measureable early event in Giant pond snails exposed to radiolysis products generated from decay of internalized/accumulated Ra-226.

Hemocyanin also displays a Bohr effect akin to that of hemoglobin that may be important to how the snail brain detects and responds to low oxygen environments and motivates individulas to move to the water/air interface and initiate pulminate breathing.

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Strategy

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For non-human biota adverse outcomes would be those outcomes that impact individual and population level risk criteria such as growth, survival and reproductive success. Since L. stagnalis is pulmonated (i.e., contains lung-like organs), when dissolved oxygen is low, it can add to its oxygen uptake via a respiratory pneumostome “hole” acting as a flow path at the air/water surface to obtain oxygen from the air [15]. The stress of low oxygen conditions, therefore, is displayed as a behavioural adaptation that enables continued growth and survival.

Hemocyanin and Radiation Effects on Oxygen Binding Capacity

The copper-containing protein hemocyanin is a major component of the snail's hemolymph involving oxygen binding and delivery to cells akin to that of mammalian hemoglobin. In the presence of calcium, the hemocyanin from L. stagnalis hemolymph cooperatively binds oxygen in the pH range 6.8-8.5 with a Hill coefficient of 3.5-4.0 and a small Bohr effect at lower pH [16] [17].  Other blue-blooded species with structurally different hemocyanin proteins complexes display different Hill coefficients for oxygen binding [Gonzalez, Nova, Del Campo et al. 2017, BBA Proteins and Proteomics].

In general, ionizing radiation lowers the oxygen binding of hemocyanins in the crab species Limulus and in welk species Busycon [18] [19] that typically have limiting G-values in irradiated oxygen-free media of 0.026 and 0.044, respectively [18], however, oxygen binding capacity can be preserved if irradiation occurs in the presense of hydroxyl radical scavengers. 

Thre is some quesiton on whether oxygen binding capacity is lost when hemocyanin is in its oxy-state or in its deoxy-state at the time of irradiation [19].  In one explanation, the effect of ionizing radiation on hemocyanin oxygen binding is thought to be due to the un-scavenged hydrated electrons produced [18], however, the levels of the radiolysis product, hydrogen peroxide, may be important [19].  Unlike what is measured for hemocyanin from Limulus, ionizing radiation has a dual effect on the hemocyanin from the welk Busycon via the protein's copper moiety: at low doses, and thus low hydrogen peroxide yield, the copper within the hemocyanin molecule becomes oxidized, thus lowering the hemocyanin oxygen binding capacity. At higher doses, the corresponding higher yields of hydrogen peroxide act to reduce the copper moiety, thus restoring the oxygen binding capacity of any hemocyanin molecules in the met‑form.

Measuring Oxygen Binding by Hemocyanin

In transitioning from its deoxygenated form to its oxygenated form, the hemocyannin protein changes from colorless to blue with an UV-visible absorption peak at ~348 nm [32] 29 . For this reason, species with hemocyanin are referred to as “blue blooded”. In its met‑hemocyanin form, the oxidized copper moiety cannot bind oxygen and its UV-visible absorption peak at 348 nm disappears, while a peak around 380-410 nm appears [29]. 

A possible early sign that internalized Ra-226 may be impacting the snail physiology is through an oxidizing effect of ionizing radiation on hemocyanin and the oxygen binding capacity of this protein. A change in oxygen binding capacity by this protein may be interpreted as a low oxygen environment, where levels of dissolved oxygen in the water medium fall below what is sufficient for cutaneous respiration. If this is the case, further biological events may manifest in affected snails by employing comparatively more pulminate breathing with increased Ra-226. Possible longer-term impacts of such events, with associated behavioural cues, would be reduced overall metabolism, slower growth and a longer time needed to reach reproductive maturity.

Summary of the AOP

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Events:

Molecular Initiating Events (MIE)
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Key Events (KE)
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Adverse Outcomes (AO)
An AO is a specialized KE that represents the end (an adverse outcome of regulatory significance) of an AOP. More help
Type Event ID Title Short name
MIE 1686 Deposition of Energy Energy Deposition
KE 1632 Increase in reactive oxygen and nitrogen species (RONS) Increase in RONS
KE 2073 increase oxidation of the di-copper moiety of the hemocyanin active site methemocyanin formation (decrease overall oxygen binding capacity)
KE 2074 Decreased, oxygen binding capacity by hemocyanin Decrease overall oxygen binding capacity (methemocyanin formation)
KE 2075 Cognitive, sensed as hypoxic/low oxygen environment Hemocyanin Bohr effect decrease
KE 2076 Increase, hemocyanin mRNA Increase, hemocyanin mRNA
KE 2077 Increase, pulmonate breathing behavioral change leading to possible reduced feeding opportunity
AO 1521 Decrease, Growth Decrease, Growth
AO 1141 Decreased, Reproductive Success Decreased, Reproductive Success

Relationships Between Two Key Events (Including MIEs and AOs)

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Network View

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Prototypical Stressors

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Life Stage Applicability

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Life stage Evidence
All life stages Not Specified

Taxonomic Applicability

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Sex Applicability

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Overall Assessment of the AOP

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Domain of Applicability

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All blue blooded species 

Essentiality of the Key Events

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Energy Deposition

Radiolysis

Oxidation of copper moieties in copper bearing proteins

Connecttion levels of biological organization  -- omics 

Changes in behaviour affecting growth/survival/reproductive success

Evidence Assessment

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Known Modulating Factors

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Modulating Factor (MF) Influence or Outcome KER(s) involved
     

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References

List of the literature that was cited for this AOP. More help
  1. Canadian Council of Ministers of the Environment, “A Protocol for the Derivation of Water Quality Guidelines for the Protection of Aquatic Life 2007”, 2007. https://ccme.ca/en/res/protocol-for-the-derivation-of-water-quality-guidelines-for-the-protection-of-aquatic-life-2007-en.pdf
  2. Fodor, I., A. Hussein, P.R. Benjamin, J.M. Koene, and Z. Pirger, “The Natural History of Model Organisms: The Unlimited Potential of the Great Pond Snail, Lymnaea Stagnalis”, eLife, 9: e56962, 2020. https://doi.org/10.7554/eLife.56962
  3. Berrie, A.D., “On the Life Cycle of Lymnaea Stagnalis (L.) in the West of Scotland”, Journal of Molluscan Studies, Vol. 36(5), pp. 283–295, 1965. https://doi.org/10.1093/oxfordjournals.mollus.a064956
  4. Boag, D.A. and P.S.M. Pearlstone, “On the Life Cycle of Lymnaea stagnalis (Pulmonata: Gastropoda) in Southwestern Alberta”, Canadian Journal of Zoology, Vol. 57(2), 1979. https://doi.org/10.1139/z79-041
  5. Hirata, A.A., “Studies on shell formation. II. A mantle-shell preparation for in vitro studies”, The Biological Bulletin, Vol. 104(3), 1953. https://doi.org/10.2307/1538492
  6. Jodrey, L.H., “Studies on Shell Formation. III. Measurement of Calcium Deposition in Shell and Calcium Turnover in Mantle Tissue Using the Mantle-Shell Preparation and Ca45”, The Biological Bulletin, Vol. 104(3), pp. 398–407, 1953. https://doi.org/10.2307/1538493
  7. Greenaway, P., “Calcium Regulation in the Freshwater Mollusc, Limnaea Stagnalis (L.) (Gastropoda: Pulmonata) : I. The Effect of Internal and External Calcium Concentration”, Journal of Experimental Biology, Vol. 54(1), pp. 199–214, 1971. https://doi.org/10.1242/jeb.54.1.199
  8. Debye, P. and E. Hückel, “The theory of electrolytes. I. Lowering of freezing point and related phenomena”, Physikalische Zeitschrift, Vol. 24, pp. 185–206, 1923.
  9. Sminia, T., N.D. de With, J.L. Bos, M.E. van Nieuwmegen, M.P. Witter, and J. Wondergem. “Structure and Function of the Calcium Cells of the Freshwater Pulmonate Snail Lymnaea Stagnalis”, Netherlands Journal of Zoology, Vol. 27(2), pp. 195–208, 1976. brill.com-previewpdf-journals-article
  10. Jeffree, R. and R.D. Simpson, “Radium-226 is Accumulated in Calcium Granules in the Tissues of the Freswater Mussel, Velesunio Angasi: Support for a Metabolic Analogue Hypothesis”, Vol. 79A(1), pp. 61-72, 1984. https://opus.lib.uts.edu.au/handle/10453/14984
  11. Greenaway, P., “Calcium Regulation in the Freshwater Mollusc Limnaea Stagnalis (L.). (Gastropoda: Pulmonata). II. Calcium Movements between Internal Calcium Compartments”, The Journal of Experimental Biology, Vol. 54(3), pp. 609–620, 1971. https://doi.org/10.1242/jeb.54.3.609
  12. Van Der Borght, O., “Accumulation of Radium-226 by the Freshwater Gastropod Lymnaea Stagnalis L.”, Nature, Vol. 197, pp. 612-613, 1963. https://doi.org/10.1038/197612a0
  13. de With, N.D., G.J. van der Wilt, and R.C. van der Schors, “Studies on the Constancy of the Value of the Ionic Product Ca2+ × CO32- in the Haemolymph of the Freshwater Snail Lymnaea Stagnalis”, in Westbroek P., de Jong E.W. (eds) Biomineralization and Biological Metal Accumulation, Springer, Dordrecht, pp. 149–153, 1983. https://doi.org/10.1007/978-94-009-7944-4_13
  14. Mozumder, A., “Chapter 2 - Interaction of Radiation with Matter: Energy Transfer from Fast Charged Particles”, Fundamentals of Radiation Chemistry, Academic Press, pp. 5-39, 1999. https://doi.org/10.1016/B978-0-12-509390-3.X5000-0
  15. Kuroda, R. and M. Abe, “The Pond Snail Lymnaea Stagnalis”, EvoDevo, Vol. 11(24), 2020. https://doi.org/10.1186/s13227-020-00169-4
  16. Dawson, A. and E.J. Wood, “Equilibrium and Kinetic Studies of Oxygen Binding to the Haemocyanin from the Freshwater Snail Lymnaea Stagnalis”, Biochemical Journal, Vol. 207(1), pp. 145–53, 1982. https://doi.org/10.1042/bj2070145
  17. Haul, R.L., J.S. Pearson, and E.J. Wood, “The Haemocyanin of Lymnaea Stagnalis L. (Gastropoda: Pulmonata)”, Comparative Biochemistry and Physiology Part B: Comparative Biochemistry, Vol. 52(2), pp. 211–218, 1975. https://doi.org/10.1016/0305-0491(75)90054-1
  18. Ke, C.H. and J. Schubert, “Radiation Chemical Studies of Hemocyanin in Oxygen-Free Media”, Radiation Research, Vol. 49(3), pp. 507–520, 1972. https://doi.org/10.2307/3573411
  19. Schubert, J. and E.R. White, “Radiation of Hemocyanin: Inactivation and Reactivation of Oxygen-Carrying Capacity”, Science, Vol. 155(3765), pp. 1000–1003, 1967. https://doi.org/10.1126/science.155.3765.1000
  20. Seppälä, O. and K. Leicht, “Activation of the immune defence of the freshwater snail Lymnaea stagnalis by different immune elicitors”, Journal of Experimental Biology, Vol. 216(15), pp. 2902–2907, 2013. https://doi.org/10.1242/jeb.084947
  21. Salo, T., C. Stamm, F.J. Burdon, K. Räsänen, and O. Seppälä, “Resilience to heat waves in the aquatic snail Lymnaea stagnalis: Additive and interactive effects with micropollutants”, Freshwater Biology, Vol. 62(11), pp. 1831–1846, 2017. https://doi.org/10.1111/fwb.12999
  22. Langeloh, L., J. Behrmann-Godel, and O. Seppälä, “Natural Selection on Immune Defense: A Field Experiment”, Evolution, Vol. 71(2), pp. 227–237, 2017. https://doi.org/10.1111/evo.13148
  23. Beltramini, M., L. Bubacco, L. Casella, G. Alzuet, M. Gullotti, and B. Salvato, “The Oxidation of Hemocyanin”, European Journal of Biochemistry, Vol. 232(1), pp. 98–105, 1995. https://doi.org/10.1111/j.1432-1033.1995.tb20786.x