Involvement of AAT transporters in methylmercury toxicity in Caenorhabditis elegans

https://doi.org/10.1016/j.bbrc.2013.04.090Get rights and content

Highlights

  • Methylmercury toxicity in worms is blocked by l-methionine.

  • Amino acid transporters transport methylmercury in the worm.

  • Knockdown of aat, increase worm the survival following methylmercury treatment.

  • Knockdown of aat also attenuate worm methylmercury content.

Abstract

Methylmercury (MeHg) is a potent neurotoxin that enters mammalian cells as a conjugate with l-cysteine through L-type large neutral amino acid transporter, LAT1, by a molecular mimicry mechanism by structurally resembling l-methionine. Caenorhabditis elegans (C. elegans) has been increasingly used to study the neurotoxic effects of MeHg, but little is known about uptake and transport of MeHg in the worm. This study examined whether MeHg uptake through LAT1 is evolutionarily conserved in nematodes. MeHg toxicity in C. elegans was blocked by pre-treatment of worms with l-methionine, suggesting a role for amino acid transporters in MeHg transport. Knockdown of aat-1, aat-2, and aat-3, worm homologues to LAT1, increased the survival of C. elegans following MeHg treatment and significantly attenuated MeHg content following exposure. These results indicate that MeHg is transported in the worm by a conserved mechanism dependent on functioning amino acid transporters.

Introduction

Methylmercury (MeHg) is a toxic heavy metal that poses a considerable risk to human health. Major sources of exposure to MeHg occur through manufacturing and consumption of seafood [5], [9]. Mass poisonings in Japan and Iraq, as well as the examination of seafood-rich diets of the Seychelles and Faroe Islands, have illustrated the effects of MeHg on human populations [8], [10]. In adults, MeHg causes focal lesions, such as loss of cerebellar granular cells and occipital lobe damage, and during extreme poisonings can lead to ataxia, numbness of extremities, muscle weakness, vision and hearing problems and paralysis [5]. As MeHg can cross the placenta, the developing fetus is also at risk; where MeHg exposure leads to microcephaly and inhibition of neuronal migration, distortion of cortical layers, cerebellar abnormalities, alterations in glial cells and alterations in neurotransmitter systems [5]. MeHg has also been implicated in neurodegenerative disorders, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis [19].

Recently the Caenorhabditis elegans (C. elegans) model has been used to characterize in vivo MeHg toxicity. C. elegans is a powerful model organism for exploring toxicity of metals due to their high homology to humans, short lifespan, ease of genetic manipulability, low cost and transparency for imaging. Many of the toxic effects of MeHg in mammals occur in C. elegans, including lethality, growth and developmental delays, and altered behavior [12], [13]. MeHg increases oxidative stress and depletes cellular glutathione levels in C. elegans, as well as induces stress response genes, such as glutathione S-transferases, heat shock proteins and γ-glutamylcysteine synthetase [12], [24]. These effects are directly related to the ability of MeHg to enter the worm. C. elegans readily accumulate Hg following MeHg exposure [13], however it is unclear how worms uptake and transport MeHg.

The molecular mechanisms responsible for the absorption and transport of MeHg are not fully characterized. While MeHg is lipid soluble and may distribute throughout the body by diffusion, it also has a high affinity for -SH (thiol) groups, forming conjugates with l-cysteine and glutathione [15]. MeHg-l-cysteine conjugates are structurally similar to l-methionine, and thus by molecular mimicry enter cells through the L-type large neutral amino acid transporter 1 (LAT1) [1], [16], [26], [32]. LAT1 is a member of the L-type Na+-independent heteromeric amino acid transporter system family, which is composed of a catalytic multi-transmembrane spanning light chain and a type II glycoprotein heavy chain complexed together by a disulfide bond [18], [22]. Substrates for transport by LAT1 include branched and aromatic amino acids, such as leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, histidine and methionine. C. elegans have nine genes encoding amino acid transporters homologous to the light chain, aat-1-9 and two genes encoding glycoprotein heavy chains, atg-1 and atg-2[31]. AAT-1 through AAT-3 have the highest homology to LAT1 and contain the cysteine responsible for the disulfide bond between heavy and light chains [31]. AAT-1 and AAT-3, but not AAT-2, have been shown to associate with ATG-2 in Xenopus oocytes and transport l-alanine [31]. However, the function of these transporters has not been characterized in the worm in vivo. AAT-4 through AAT-9 have the least homology to human LAT1 and do not contain the cysteine residue required for bonding with the heavy chain [30]. Recently it has been reported that AAT-6 interacts with the scaffold protein Na+/H+ exchanger regulatory factor, NRFL-1, to localize to the membrane [11], but it has not been reported whether NRFL-1 is required for other AAT proteins as well. Herein we examined the hypothesis that MeHg is transported in C. elegans by a mechanism similar to mammals: entering cells through AAT transporters as a cysteine conjugate, which may be competitively blocked by excess l-methionine. Additionally we examined whether NRFL-1 was involved in MeHg-induced toxicity.

Section snippets

Reagents

Unless otherwise stated all reagents were obtained from Sigma–Aldrich (St. Louis, MO).

C. elegans strains and handling of the worms

C. elegans strains were handled and maintained at 20 °C on Nematode Growth Medium (NGM) plates seeded with OP-50 strain of Escherichia coli, as previously described [3]. The following strains were used in this study: N2 and NL2099 (rrf-3(pk1426)). All strains were provided by the Caenorhabditis Genetic Center (CGC; University of Minnesota). Synchronous L1 populations were obtained by isolating embryos from

l-methionine protects worms against MeHg-induced lethality

In mammals, MeHg is transported as a l-cysteine conjugate by amino acid transporters, however it is unknown whether there are additional transport mechanisms or how MeHg is transported in invertebrates. To address whether a shared mechanism for MeHg transport via molecular mimicry occurs in worms, C. elegans were pre-treated with 1 mM l-methionine, to compete as a substrate for amino acid transporters. Pre-treated and untreated worms were then treated with increasing concentrations of MeHg (10 μM

Discussion

Caenorhabditis elegans is an emerging model organism for MeHg toxicity research [12], [13], [24], [29], however little is known about how worms uptake or transport MeHg. As C. elegans contain 60–80% gene homology with mammals [17], [23], examining MeHg transport in nematodes can provide further insight on both known and unknown transport mechanisms. The present study provides direct evidence for an evolutionarily conserved transport mechanism for MeHg via LAT1 homologues in C. elegans.

Amino

Acknowledgment

This work was funded by NIEHS R01 ES007331 and S10 RR026742.

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