Research article
Examining the effect of manganese on physiological processes: Invertebrate models

https://doi.org/10.1016/j.cbpc.2021.109209Get rights and content

Highlights

  • Exposure to Mn2+ slows development and decreases survival of Drosophila larvae.

  • Mn2+ depresses cardiac function in larval Drosophila.

  • Synaptic transmission at neuromuscular junctions in Drosophila and crayfish is depressed by Mn2+.

  • Proprioceptive function in crab limbs is compromised by Mn2+ in a dose-dependent manner.

Abstract

Manganese (Mn2+ as MnSO4 &/or MnCl2) is a common and essential element for maintaining life in plants and animals and is found in soil, fresh waters and marine waters; however, over exposure is toxic to organisms. MnSO4 is added to soil for agricultural purposes and people are exposed to Mn2+ in the mining industry. Hypermanganesemia in mammals is associated with neurological issues mimicking Parkinson's disease (PD) and appears to target dopaminergic neural circuits. However, it also seems that hypermanganesemia can affect many aspects of health besides dopaminergic synapses. We examined the effect on development, behavior, survival, cardiac function, and glutamatergic synaptic transmission in the Drosophila melanogaster. In addition, we examined the effect of Mn2+ on a sensory proprioceptive organ and nerve conduction in a marine crustacean and synaptic transmission at glutamatergic neuromuscular junctions of freshwater crayfish. A dose-response effect of higher Mn2+ retards development, survival and cardiac function in larval Drosophila and survival in larvae and adults. MnSO4 as well as MnCl2 blocks stretch activated responses in primary proprioceptive neurons in a dose-response manner. Mn2+ blocks glutamatergic synaptic transmission in Drosophila as well as crayfish via presynaptic action. This study is relevant in demonstrating the effects of Mn2+ on various physiological functions in order to learn more about acute and long-term consequences Mn2+ exposure.

Introduction

Given that manganese (Mn2+) is found in many plants and animals that are consumed by humans as food, it is essential to understand the effects of consuming high levels of Mn. It is well established as an essential element required for photosynthetic abilities in plants as well as cellular function in mammals. For example, Mn acts as a cofactor for aiding in the function of various essential enzymes (i.e., superoxide dismutase) in organisms. However, Mn is also toxic at high levels. Thus, it is of interest to understand the threshold at which subtle effects in physiological processes become compromised. It is difficult to determine what physiological symptoms may manifest when chronically exposed to low levels of manganese but examining acute actions at high levels may provide some insight to what the potential effects might be. Mn is found in seaweed (130 to 735 mg/kg dry weight), formula milk consumed by developing infants (above recommended daily limits; Frisbie et al., 2019), marine and freshwater fish (<0.2 to 19 mg/kg dry weight) and can bioaccumulate in blueberries as well is found on plants from application of agricultural sprays in the range of 2000–4000 mg/kg (Howe et al., 2004).

Hypermanganesemia in mammals can be toxic and is associated with neurological issues mimicking Parkinson's disease (PD). In fact, the neural injury from Mn affects the same locations within the central nervous system known to be dysfunctional in PD. Manganese has been shown to accumulate in these areas (Calne et al., 1994; Eriksson et al., 1987; Erikson et al., 2004a, Erikson et al., 2004; Brenneman et al., 1999; Nagatomo et al., 1999; Newland, 1999; Pal et al., 1999; Baek et al., 2003; Nelson et al., 2018). Such findings have led, in part, to suggesting that Mn selectively targets dopaminergic neurons. However, it is not known what the effect(s) are of Mn on neurons which use other types of neurotransmitters or exactly how Mn2+ is offsetting the dopaminergic neural circuit in the mammalian brain. Bonilla-Ramirez et al. (2011) addressed whether Mn was accumulating in the dopaminergic regions of the adult brain of D. melanogaster and found that there indeed was an accumulation and that there was a dose-dependent effect on the adults being able to locomote in a climbing assay. Additionally, acute and long-term feeding of MnSO4 decreased survival of the adult Drosophila likely due to damage in the dopaminergic regions within the CNS.

Nelson et al. (2018) reported on studies conducted with bivalve mollusks where the cilia beating is controlled by dopaminergic and serotonergic innervation. Low concentration of Mn2+ at 50 μM reduced the dopaminergic effects on the cilia beating but a higher concentration of Mn2+ blocked the effect with application of DA. This implied that low concentrations reduced the neuron's function, potentially by a decrease in DA release but that high concentrations blocked the postsynaptic DA response (Catapane et al., 1978; Aiello, 1990; Carroll and Catapane, 2007; Martin et al., 2008; Nelson et al., 2010, Nelson et al., 2018). Nelson et al. (2018) investigated the mechanism in which Mn2+ altered the postsynaptic response. This previous study focused to address if Mn2+ blocked the DA receptors as well as the downstream effects of adenylyl cyclase for the G protein coupled metabotropic receptors (GPCR) of DA. It appeared that Mn2+ resulted in a reduced sensitivity to DA by likely desensitizing the DA receptors and not acutely altering adenylyl cyclase (Nelson et al., 2018). The notion in the effect of Mn exposure is that oxidative stress and altered mitochondrial function occurs (Bowman et al., 2011; Nelson et al., 2018), resulting in the neurons becoming dysfunctional. Such cellular mechanisms would likely be affected in all neurons and other cell types as well. There are many potential actions on neurons by affecting channels and screening electrical potential on membranes. To gain an overview in the effects of Mn2+ on various physiological functions, we conducted experiments in a few model systems in different animals on development, survival, cardiac, sensory, motor, and glutamate synaptic responses.

Larval D. melanogaster have a relatively simple neural circuit to analyze metal toxicity effects on locomotive behaviors. Body wall crawling, and mouth hook movements are readily assessed behaviors to examine the dietary actions of consuming Mn2+ (Badre and Cooper, 2008; Dasari et al., 2007). Thus, to gain some insight into the effects of dietary Mn2+, these larval behaviors were examined in this study. As expected, if the neural circuits for eating and locomotion are severely affected in adults and larvae, one would expect manganese to also affect development and survival. Thus, the general effects on the pupation rate as well as survival of larvae and adults with manganese-tainted food was surveyed herein.

The direct effect of Mn2+ on sensory neurons is lacking in literature. Since Mn2+ can block voltage-gated calcium channels, then it may be possible that Mn2+ can block stretch-activated channels (SACs). We examined the effects of Mn2+ exposure on primary sensory neurons activated by SACs in proprioceptive neurons of a model crab chordotonal organ (Alexandrowicz, 1972; Cooper, 2008; Hartman and Boettiger, 1967; Whitear, 1938).

To address whether Mn2+ has an effect beyond that on dopaminergic synapses, evoked and quantal responses of glutamatergic synapses were also observed when acutely exposed to Mn2+. The spontaneous quantal events of larval D. melanogaster and crayfish neuromuscular junctions (NMJs) were used to assess the acute actions on the model glutamatergic synapse (Titlow and Cooper, 2018). If there remains the presence of spontaneous quantal events of the same general magnitude before and during exposure to Mn2+ while evoked responses are retarded, then these effects would indicate a presynaptic action; however, if both evoked and spontaneous quantal events were gradually declining in amplitude with exposure, then it would be suggestive that the glutamate receptors are affected by Mn2+.

In rodent models, Mn2+ exposure (1–8 mM) was found to inhibit myocardial contraction and electrical function (Jiang and Zheng, 2005). Thus, we examined the acute effect on the heart rate in larval D. melanogaster within in situ preparations. When the in situ larval hearts are bathed in a large amount of saline, leaving them isolated from any endogenous hormonal actions, then the direct effects of exogenously applied compounds can be assessed independently of endogenous compounds of the hemolymph. The heart rate is rapidly altered with release of dopamine, serotonin or octopamine by the larval CNS, (Malloy et al., 2017) which is not an issue with saline flushed in situ preparation of the larval heart. Since the D. melanogaster model is increasingly becoming a model to address cardiac function and pacemaker activity, it serves as a rapid approach to screen the effect of Mn2+ on cardiac function. Since the larval heart tube is not innervated until the transition from larvae to pupa, the direct action on the heart is able to be addressed independently of neural innervation (Johnstone and Cooper, 2006). The heart rate in larval D. melanogaster is very sensitive to extracellular Ca2+ levels, pH, and other modulators, so the change in heart rate can be used as a sensitive index for the effects of compounds that alter these functions (de Castro et al., 2014; Desai-Shah et al., 2010; Majeed et al., 2013, Majeed et al., 2014; Titlow et al., 2013). Thus, it was assumed that there would likely be an acute effect by Mn2+ on the heart rate in larval Drosophila due to the responsiveness to Ca2+ in bathing media.

The common forms of Mn in nature are found in the oxidation state of +2 (e.g., manganese chloride [MnCl2]), +4 (e.g., manganese dioxide [MnO2]), and + 7 (e.g., potassium permanganate [KMnO4]) (Howe et al., 2004). Both MnSO4 and MnCl2 are both readily soluble in water and readily dissociate in water. Since cytotoxicity varies between MnSO4 and MnCl2 in some studies (Marreilha dos Santos et al., 2008), we examined the two compounds in some studies herein. The differences in the physiological responses between the compounds appear to be due to differences in the rate of transport into different tissues (Erikson et al., 2004a, Erikson et al., 2004). In the current study, some of the physiological assays were performed with MnSO4 and MnCl2 either being dissolved in food or in the saline applied to exposed tissues.

Surveying several physiological systems in various animals will provide a general understanding of the effect of Mn2+. This knowledge can then be applied to understand the effects on organisms potentially exposed to Mn2+ through industrial mining or agricultural use of MnSO4 in folate sprays intended to benefit plant health. Mn2+ is noted to be found in various tissues of the edible blue crab in marine environments (Callinectes sapidus) (Zotti et al., 2016) and edible crayfish of freshwater systems. Given that MnSO4 is used in agriculture to aid plant health, it is also found on food sources for insects not only in the soil but also in and on plants (Chen et al., 2020; Jhanji et al., 2014). Increasing manganese concentrations also seems to be linked to idiopathic blindness in the American lobster, a condition affecting roughly half of the species population (Ochs et al., 2020). Additionally, commonly eaten crabs in the wetlands of China were found to contain high amounts of manganese that deemed them to be inedible (Zhang et al., 2019). Manganese can be taken up across the gills of crustaceans and is found in the hemolymph (7.35 μg/g wet weight) as well as the midgut of hermit crab (1596 μg/g dw) and in crayfish (374 μg/g dw). In skeletal muscle of crayfish it has been found to be as high as 87 μg/g and in the central nervous system in lobster as high as 193 μg/g. Baden and Eriksson (2006) and Zhang et al. (2019) reviewed the bioaccumulation in various tissues of crustaceans under different conditions. A detailed review of accumulation of Mn2+ in tissues of eatable lobster was reviewed by Ochs et al. (2020). Certain edible termites have also been found to contain astonishingly high amounts of Mn2+, some species containing enough that minimal consumption could exceed the upper limits for intake of Mn2+ in humans (Verspoor et al., 2020).

Because of the Mn2+ exposure on various populations in the ecosystem examining the effects on D. melanogaster can provide an index (Mohandas et al., 2017) of the potential effects on other insect species in the environment. As it is well established, the effects of agents on non-humans are relevant to understanding the potential actions of that agent in humans. To uncover the potential effects of compounds on physiological functions, a higher concentration than that which a system is normally exposed to can unmask the more subtle effects of the compound present at a lower level over longer period of time. Since Mn is of higher concentration in some infant formula products, there is some interest in understanding the potential effects of Mn2+ on neuronal function of human infants fed formula milk in comparison to those fed human breast milk (Frisbie et al., 2019). Thus, with all of these potential avenues for Mn exposure, it is crucial to understand more about the effects of manganese in various other organisms in order to better predict the potential effects on humans.

Section snippets

Animals

Drosophila melanogaster, Canton S (CS) flies were used in all behavioral and physiological assays. This strain has been isogenic in the lab for several years and was originally obtained from Bloomington Drosophila Stock Center (BDSC). All animals were maintained in vials partially filled with a cornmeal-agar-dextrose-yeast medium. Blue crabs (C. sapidus) were obtained from a local supermarket in Lexington, KY, USA which were delivered from a distribution center in Atlanta, GA, USA. They were

Survival and developmental studies of Drosophila melanogaster

Larval development and survival study were conducted in triplicate with ten 1st instars, providing a total of 30 individuals for each condition. Within a 6 h window from hatching, the 1st instars were placed in food tainted with NaCl, MnSO4 and MnCl2 at various concentrations (0.0025, 0.015, 0.03 and 0.1 M). The most significant effect was for the higher concentrations of 0.03 and 0.01 M MnSO4 and MnCl2 as this was toxic for the 1st instar. In these conditions, there were no 2nd instars

Discussion

In this study, we have shown that Mn2+ from MnCl2 or MnSO4 can have profound effects in a dose-dependent manner on development, survival of larvae and adults, behaviors, and cardiac function of D. melanogaster. In addition, Mn2+ depresses synaptic transmission at the D. melanogaster and crayfish NMJs with the proposed mechanism being the blockage of evoked presynaptic transmission. The presence of spontaneous quantal events indicates that the sensitivity of the postsynaptic glutamate receptors

Funding

Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103436 (H.T.). and Howard Hughes Medical Institute (#52008116) awarded to the Univ. KY (V.M. Cassone, PI) for development of StemCats undergraduate research experiences. Chellgren Endowed Funding (R.L.C.).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References (60)

  • A.P. Marreilha dos Santos et al.

    Antioxidants prevent the cytotoxicity of manganese in RBE4 cells

    Brain Res.

    (2008)
  • K. Martin et al.

    The neurotoxic effects of manganese on the dopaminergic innervation of the gill of the bivalve mollusc Crassostrea virginica

    (2008)
  • S. McCubbin et al.

    Pharmacological profiling of stretch activated channels in proprioceptive neuron

    Comp. Biochem. Physiol. C

    (2020)
  • G. Mohandas et al.

    Whey protein isolate enrichment attenuates manganese-induced oxidative stress and neurotoxicity in Drosophila melanogaster: relevance to Parkinson's disease

    Biomed. Pharmacother.

    (2017)
  • S. Nagatomo et al.

    Manganese intoxication during total parenteral nutrition: report of two cases and review of the literature

    J. Neurol. Sci.

    (1999)
  • M. Nelson et al.

    Effects of p-aminosalicylic acid on the neurotoxicity of manganese on the dopaminergic innervation of the cilia of the lateral cells of the gill of the bivalve mollusc Crassostrea virginica

    Effects of p-aminosalicylic acid on the neurotoxicity of manganese on the dopaminergic innervation of the cilia of the lateral cells of the gill of the bivalve mollusc Crassostrea virginica

    (2010)
  • M. Nelson et al.

    Manganese toxicity is targeting an early step in the dopamine signal transduction pathway that controls lateral cilia activity in the bivalve mollusc Crassostrea virginica

    Comp. Biochem. Physiol. C

    (2018)
  • A.T. Ochs et al.

    Acute and long-term manganese exposure and subsequent accumulation in relation to idiopathic blindness in the American lobster, Homarus americanus

    Aqua. Toxicol.

    (2020)
  • N. Tanabe et al.

    Transmitter release at frog end-plate loaded with a Ca2+-chelator, BAPTA: hypertonicity and erythrosin B augment the release independently of internal Ca2+

    Neurosci. Let.

    (1988)
  • Z. Zhang et al.

    Copper, zinc, manganese, cadmium and chromium in crabs from the mangrove wetlands in Qi'ao island, South China: levels, bioaccumulation and dietary exposure

    Watershed Ecol. Environ.

    (2019)
  • M. Zotti et al.

    Comparative analysis of the proximate and elemental composition of the blue crab Callinectes sapidus, the warty crab Eriphia verrucosa, and the edible crab Cancer pagurus

    Heliyon.

    (2016)
  • E.L. Aiello

    Nervous control of gill ciliary activity in Mytilus edulis

  • J.S. Alexandrowicz

    The comparative anatomy of leg propriocetors in some decapod crustacea

    J. Mar. Biol. Assoc. UK

    (1972)
  • S.P. Baden et al.

    Role, routes and effects of manganese in crustaceans

    Oceanogr. Mar. Biol.

    (2006)
  • N.H. Badre et al.

    Reduced calcium channel function in Drosophila disrupts associative learning in larva, and behavior in adults

    Inter. J. Zool. Res.

    (2008)
  • L. Bonilla-Ramirez et al.

    Acute and chronic metal exposure impairs locomotion activity in Drosophila melanogaster: a model to study Parkinsonism

    Biometals.

    (2011)
  • K.A. Brenneman et al.

    Manganese induced developmental neurotoxicity in the CD rat: is oxidative damage a mechanism of action?

    Neurotoxicology

    (1999)
  • D.B. Calne et al.

    Manganism and idiopathic parkinsonism: similarities and differences

    Neurology

    (1994)
  • E.J. Catapane et al.

    Pharmacological study of the reciprocal dual innervation of the lateral ciliated gill epithelium by the CNS of Mytilus edulis (Bivalvia)

    J. Exp. Biol.

    (1978)
  • R.L. Cooper

    Proprioceptive neurons of chordotonal organs in the crab, Cancer magister Dana (Decapoda, Brachyura)

    Crustaceana

    (2008)
  • Cited by (0)

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