Metallothioneins in Earthworms: The Journey So Far

The soil is a major repository for contamination where terrestrial organisms are exposed to pollution. Earthworms are important organism in the terrestrial ecosystem and their ecological functions are indispensable as they participate in various processes in soil. They form signifi cant biomass in the terrestrial ecosystem and they occupy a sensitive position in the food chain. Lumbricus terrestris, L. rubellus, Eisenia fetida and E. andrei are relevant earthworm species for monitoring environmental pollution [1] in terrestrial ecotoxicology studies. This is attributed to their capability to accumulate and tolerate elevated amounts of toxic metals within a certain threshold without experiencing signifi cant damages [2]. Their survival and tolerance are dependent on the regulation/excretion of metallic trace elements and detoxifi cation of non-essential toxic metal ions.


Introduction
The soil is a major repository for contamination where terrestrial organisms are exposed to pollution. Earthworms are important organism in the terrestrial ecosystem and their ecological functions are indispensable as they participate in various processes in soil. They form signifi cant biomass in the terrestrial ecosystem and they occupy a sensitive position in the food chain. Lumbricus terrestris, L. rubellus, Eisenia fetida and E. andrei are relevant earthworm species for monitoring environmental pollution [1] in terrestrial ecotoxicology studies. This is attributed to their capability to accumulate and tolerate elevated amounts of toxic metals within a certain threshold without experiencing signifi cant damages [2]. Their survival and tolerance are dependent on the regulation/excretion of metallic trace elements and detoxifi cation of non-essential toxic metal ions.
Earthworms are affected by soil contaminants at the various levels of biological organization from sub-organismal, individual to population levels. The passageways of contact with contaminants are majorly through the skin from the interstitial pore or from the ingestion of soil particles into their guts [3]. In their adaptive responses to such environmental stress, they exhibit non-transferable physiological adaptations which could induce metabolic modifi cations making them more tolerable to such environmental changes [4] like metal contamination [5] and [6]. On the other hand, the coping mechanisms could involve changes that would be transferable to offspring hence forming ecotypes of earthworm species based on location found [7].
There are standardized protocols for earthworm acute and sublethal testings of chemicals in contaminated soils [1] based on their responses and behavioural patterns [9]. Advances in molecular biology make use of biomarkers as rapid diagnostic and predictive tools in environmental assessments [10]. The use of genetic biomarkers gives better insight into ecotoxicological assessments as gene expression underscores changes in functionality at all levels of organizations and the predictive effect on the ecosystem. A protocol developed from a target gene can be extrapolated and used for similar genes in other related organisms [11]; hence this approach is more reliable than conventional earthworm testings [12]. Molecular markers are generally used because they typically indicate the susceptibility of organisms to contaminants or stressors. The molecular biomarkers monitored in earthworm ecotoxicological studies include Carboxylesterase (CES), Acetylcholinesterase (AChE), Catalase (CAT) and Glutathione S Transferase (GST) activity, the concentration of glutathione (GSH), [13]. Other genetic markers used in such studies are metallothioneins, annetocin [14]. Their presence and levels in organisms are Citation: Aemere  indicative of tolerance to metal, stress and other physiological forms of pollution hence their suitability as biomarkers and indicators of environmental status and pollution.
Metallothioneins (MTs) are genetic biomarkers commonly monitored in annelids as it is referred to as the best-known biomarker candidate among Oligochaeta Annelida [15]. The common earthworm MT isoforms reported in works of literature are wMT 1 and wMT 2 [16], their induction when exposed to stress and contaminants, mechanisms of action in response to metals and their affi nity to metals are reasonably investigated. However, a third isoform is wMT 3 detected at the embryonic stage of earthworms [17]; except this report, there is no other known report on wMT 3 . Its structure and distinct mechanism of action remain obscure. This works compares the roles of the wMTs along with their mechanisms of action as well as highlighting signifi cant milestones in the progressive investigation of earthworm MTs. This work also centers on the detection of MTs in earthworms and their limitations with emphasis on the new technologies. We also reviewed the reports on and highlighted pitfalls in environmental monitoring of metallothioneins in earthworms exposed to Contaminants of Emerging Concerns (CECs) and crises of new technologies like nanotechnology on earthworm MTs.

Metallothioneins
The fi rst metallothionein was identifi ed by Margoshes and Vallee [18] and a myriad of research followed with a focus on vertebrate and mammalian isoforms [19] and [20]. Their roles in the medical fi eld are well reported [21] and [22]. Since its fi rst report, more than 11500 articles on metallothioneins are cited in PubMed, and about one-tenth of these are related to environmental studies. MTs are low molecular weight cysteine-rich (up to 33% by composition) ubiquitous proteins expressed by organisms under stress condition especially when induced by metals at certain levels, making them very well-studied targets. They are heat-stable [23] and have approximately 70 amino acids [24,25]. MTs are encoded by a multigene family which vary in their responses to different inducers including heavy metals, glucocorticoids, hormones, oxidants, strenuous exercise, superoxide and hydroxyl radicals generated by gamma radiation and cold exposure [26]. The major roles of MTs include the homeostasis of trace metals (Zn, Cu, Mn, Fe etc), protection against oxidative stress and detoxifi cation of xenobiotic metals (Pd, Cd etc) [27] and [28], metal ion transport, maintaining redox pool, scavenging of radicals and regulation of expression as explained and depicted in Figure 1 [29]. They are found in a range of organisms from microbes to mammals and reports on invertebrate MTs include nematodes [30], annelids [31]; insects [32]; the oysters [33] and various species of gastropods [34,35].
MTs have shown functional variability among organisms and signifi cant sequence heterogeneity [36] between taxa but notable conserved regions within phylogenetically related taxa [30]. Extensive reports on their detection, roles, mechanisms of action and stoichiometry in a wide variety of organisms [37] are available.

The basic structure of metallothioneins
The structures of proteins depict their functionalities. Metallothionein has a chemical confi guration often occurring as a straight polypeptide chain of cysteine (cys-cys) or cysteine having other amino acids within the chain (cys -x -cys) [38]. This makes it form better binding cluster since cysteine possesses the thiolate -SH end for metal attachment [39]. Individual cysteine residue required for metal ion binding is typically insuffi cient, hence the cluster forming tetrahedral binding arrangement using bridging sulphur binding ligands. The sulphur groups of cysteines are usually positioned adjacent to themselves hence encouraging the clustering.
Their chemical confi gurations of various families of MTs are reported but the 3D depictions are scarce [40]. Although there are structural diversity among MTs in organisms, the functional domains (C-and N-) for metal binding is usually common, appearing as "dumbbell". The functional domains only form 3D structures upon metal coordination, and when there are no metal ions, (apo-thionein or apo-T), the domains usually appear unstructured; their structure depicts their functionality [41]. One elucidated Mt 3D structure is the mammalian MTs; they have two metal-binding domains that form metal-cysteine clusters at the N-and C terminals [42]. They have structures confi gured to form folded metal-binding domains with the -domain closer to C-terminal and more stable while the other is a more reactive -domain, which is closer to N-terminal. The metal clusters formed are named "M4Cys11 (-domain) and M3Cys9 (-domain)" where M represents a divalent metal ion like Zn2+ or Cd2+ [43]. The functional domains are linked with varying lengths of amino acid sequences; these linkers determine the structural stability of the MT.

Earthworm metallothioneins structure
The mechanisms of tolerance of earthworms to metal by accumulation are attributed to expression of MTs and their formation of metal-rich granules (MRGs) [44]. Metal toxicity will only occur when the capacity of these mechanisms to bind metals is exceeded [45]. Unlike vertebrate MTs where similarities occur structurally, invertebrates MTs show inter / intra -structural diversities hence they have distant phylogenic relationships. This diversity could be due to their evolutionary changes in adaptation to their environment, which constantly predisposes them to contaminants.
MTs do exist in homologues and are referred to as isoforms in the literature [46]; invertebrates like snails and earthworms have eight and three MT isoforms respectively. The mostreported earthworm MT isoform are wMT1 and wMT2. They are often described to have a reverse mammalian MT arrangement [42] arrangement depicted in Figure 2. These two isoforms (wMT1 and wMT2) have greater than 75% similarities in their sequences but differ considerably in the length and composition of their linker sequences. wMT1 have longer linker regions (6 residues), and it is less stable than wMT2 with shorter linker sequences (4 residues) wMT2 has shown more stability in its metal retention with a wider range of pH and its effectiveness in cadmium toxicity protection than wMT1 [47].

Induction of metallothioneins in earthworms
Ecotoxicology studies involving earthworms earlier attributed major forms of cellular management of excess heavy metal to the possession of chloragosomes [48,49]. Figure 3 depicts a conceptual model of impacts on soil metal chemistry due to exposure of earthworms to metal contaminated soils.
One of the tolerance mechanisms of genetic origin is the induction of metallothionein and it is reported in several earthworm species. They include E. fetida [50,51], E. Andrei, [52,53] and Libyodrilus violaceous [54].The genetic origin of resistance is attributed to evolutionary changes in MT gene and researches suggest that MTs are the basis of metal resistance and tolerance in these organisms [55]. Earthworm MTs mainly function in metal detoxifi cation and evidence indicate that. Studies had shown metallothionein induction and their regulation in insects and vertebrates were conserved [57,58], it involved the binding of metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) usually found in the MT genes promoter. It was however established that the transcriptional activation of MTs in invertebrate is not consistent with that of the insects and invertebrates [59] but the exact mechanism is unclear. Instead, MREs were found in the invertebrate MT gene promoters in Lumbricus rubellus [60] and cAMP responsive element (CRE) was found to be involved in Cd-induced Wmt2 transcription and acted as a transcriptional activator of invertebrate MTs. Metallothionein as biomarker are monitored in earthworms for Cd contamination [61,62] and other metals like mercury and CuSO 4 [63,64] and metallothionein monitoring in earthworm ecotoxicological studies is common.

Earthworm metallothioneins induction by metals
In earthworms, metallothionein induction of two metal responsive proteins is known. They have nucleotide and amino acid sequences similarities of 80.9% and 74.7%, respectively but a distinctive deletion/insertion of two amino acids [65]. Their coding regions show a conserved arrangement of the cysteine residues which lack aromatic amino acids. The sequences of the two isoforms (wMT1 and wMT2) are structurally similar to other invertebrate MTs. The Metallothionein gene, Wmt2, is known to express the most responsive protein among wMTs.
wMT3 is a third isoform of earthworm metallothioneins derived from an EST library generated from developing cocoon and highly expressed in embryonic development. It is 67% similar and 56% identical with wMT1 and wMT2 however, their role remains unclear. The three wMTs isoforms differ in their expression patterns and levels when exposed to metal ions.   stoichiometry and protein folding of Zn-wMT2 and Cd-wMT2, conferring wMT2 its function in Cd accumulation [46].
Though, Zn (II), Cd (II), or Cu (I) are metal ions known to form metallothionein clusters, the report on their overall affi nity are species dependent. The study of Foster and Robinson [66] reported HpMTs affi nity as Cu(I) > Cd(II) > Zn(II) and Cu(I) being the most competitive ion. The study indicated that the selection and discrimination of metals by metallothioneins was not entirely based on overall affi nity instead on the interplay of other factors. wMTs preference for metals is reported in some investigations but show inconsistences; this remains an area that require further explication.

Earthworm metallothionein induction by Contaminants of Emerging Concerns (CECs)
Substances other than metals are known contaminants found in the environment where they cause detrimental effects on the biota, among such are organic secretions like toxins and drugs, especially antibiotics; they are grouped as CECs. Investigating metallothionein induction due to these contaminants is of interest in recent times and a few reports are available. van OmmenKloeke [67] reported expression of MTs in E. Andrei induced by low concentrations of 2-phenylethylisothiocyanates (ITCs), a known natural toxin. MT was recommended as an early biomarker of ITCs contamination even at low concentrations. Colistin is a feed additive used by animal farmers as antibiotics and nutrient enhancer [68]. Its suppression of MTs is shown by Guo, et al. [69] and they indicated that colistin in soils interfered with other molecular markers in metal ecotoxicity study, but MT served as early biomarker for colistin contamination. Enrofl oxacin is another antibiotic used in veterinary but did not induce MT in E. fetida [70].  [73,74]. Global production of nanoparticles is projected to increase hence the usage and disposal of these materials will be enormous. Commonly used nanoparticles include AgNPs, CoNPsCuNPs, ZnNPs and AuNPs. Study on their environmental impact is of necessity, especially in the soil ecosystem where they are subject to transformations, aggregation/agglomeration and reaction with other biomolecules, exchange of surface elements and other redox reactions [74]. These properties make them behave differently with living organisms with respect to their parent metal.
Just a handful of investigations involve MTs' use as biomarkers in nano -related ecotoxicological studies with few focusing on the detection and quantifi cation of metallothioneins in earthworms. Inductions of MT in earthworms are recorded in recent studies of Unrine, et al. [77,78]. Other such investigations include Enchytraeus crypticus exposed to AgNP [79], Lumbricus rubellus and their coelomocytes impacted by AgNPs (NM-300 K) [80] and AgNPs exposure to E. fetida causing transcriptional expression of MT [81]. The presence of nanoparticles, drugs and toxins in the environment and their impact are areas of interest in recent time, such studies involving earthworm MTs are under reported hence more investigations in this area are encouraged.

Methods of Metallothionein (Mts) Detection and Quantifi cation
The earliest detection of organic substances like cystine was by Heyrousky polarography [82] while the fi rst detection of metallothioneins was by Differential pulse polarography -DPP method [83]. In the earlier approach, cystine was the only amino acid that showed a polarographic reaction in a solution of ammonium chloride, ammonia and cobaltous chloride (Brdicka electrolyte). Conversely, cysteine and other thioacids act catalytically in the Brdcka solution which they owe to their sulfhydryl groups, the technique involves the catalysis of hydrogen in the presence of a protein containing SH-groups. Using this technique, the quantifi cation of cysteine and others were reported by Brdicka [84,85] hence the subsequent use of the term "Bridcka reaction" by Thompson and Cosson [83]. With this method, Cystine and cysteine were quantifi ed in pure solutions and hydrolysates of organic substances in work by Stern, et al. [82]. Several efforts have been made in the modifi cation of DPP technique which had yielded better results like better detection limits, rapid assays, increased sensitivity etc [86]. of the fi rst MTs [18]. Brdicka reaction (with several modifi cations -AdTS, AdTS CV, AdTS DPV) was commonly used in metallothionein detection and quantifi cation in various organisms [87,88]. Other MT detection involved using metal saturation assays in monitoring Mt in fi sh [89] and terrestrial organisms [90]. The method involves equating the quantity of MTs as a total saturation of their sulphydryl groups by metal ions. This estimation was misleading as other metal-binding ligands also exist in these biological systems could interfere with the estimation [91,92].
The present-day technique used in the detection and quantifi cation of MTs range from electrochemical to bioanalytical and molecular methods. These methods involve procedures like ELISA, enzyme-linked assays, chromatography, electrophoresis, mass spectrometry, inductive coupled plasma mass spectrometry, electrochemistry, etc. Most of these techniques, however, do have their pros and cons. The immunochemical technique was the most commonly reported in publications in metallothionein detection between 2001 and 2010 [29], it is specifi c and sensitive however limited by the diffi culty to obtain MTs antibodies among other disadvantages [93]. The electrochemical techniques like AdTS, AdTS CV, AdTS DPV [94,95] were sensitive and could detect MT peaks but require the use of analyser such as AUTOLAB Analyzer [24].
The improvement of fl uorescent technique for MT detection resulted in detecting trace amount of MTs where fl uorescent agents like ammonium-7fl uorobenzo-2-oxa-1, 3-diazole-4-sulfonate (SBD-F) [96] and monobromobimane (mBBr) [97] are derived. Geng, et al. [98] further improved on the fl uorimetric method for MT quantifi cation; it was sensitive to a wide range of MT concentrations and gave a relatively accurate estimation of MT. It however required tandem column system to separate the derived compound to eliminate interference or require a prior MT purifi cation before derivation. Also, improved colorimetric method for detecting metallothioneins (MTs) was developed by Qian, et al. [99]. It involves using a thymine (T)-rich oligonucleotide (TRO)-Hg-AuNP system. The thiol groups of MTs could interact with mercury from the T-Hg2+-T complex to release TRO, resulting in a colour change of the system. MTs concentration of the range 2.56 x108 to 3.08 x 107 mol/L and the detection limit of 7.67 x 109 mol/ L were possible. This method allows direct analysis of the samples by the naked eye without costly instruments, and it is reliable, inexpensive, and sensitive.
The advent of high-performance liquid-phase chromatography-electrospray tandem mass spectrometry (HPLC ESI MS) and high-performance liquid chromatographyinductively coupled plasma-mass spectrometry (HPLC-ICP-MS) promised more accurate quantifi cation of metallothioneins. The high costs and technicalities of this equipment remain an imperative factor to consider in their use for the advancement of biological research. Biomolecular method, e.g. ELISA, MT -mRNA (PCR and QT-PCR) are standard method used in detecting and quantifying of metallothioneins; they are simple, less technical and accessible. They can be used to distinguish Mt-isoforms but the mRNA concentration does not give an accurate estimate of the protein concentration [93].
The fi rst detection of earthworm MTs reported in 1998 [16] required the combination of gel chromatographic techniques and "novel" molecular methodologies (Directed Differential