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Chen, R.;  Tu, H.;  Chen, T. Engineered Bacteria in the Detoxification of Heavy Metals. Encyclopedia. Available online: (accessed on 09 December 2023).
Chen R,  Tu H,  Chen T. Engineered Bacteria in the Detoxification of Heavy Metals. Encyclopedia. Available at: Accessed December 09, 2023.
Chen, Runqiu, Huaijun Tu, Tingtao Chen. "Engineered Bacteria in the Detoxification of Heavy Metals" Encyclopedia, (accessed December 09, 2023).
Chen, R.,  Tu, H., & Chen, T.(2022, July 11). Engineered Bacteria in the Detoxification of Heavy Metals. In Encyclopedia.
Chen, Runqiu, et al. "Engineered Bacteria in the Detoxification of Heavy Metals." Encyclopedia. Web. 11 July, 2022.
Engineered Bacteria in the Detoxification of Heavy Metals

Heavy metal (HM) exposure remains a global occupational and environmental problem that creates a hazard to general health. Even low-level exposure to toxic metals contributes to the pathogenesis of various metabolic and immunological diseases, whereas, in this process, the gut microbiota serves as a major target and mediator of HM bioavailability and toxicity. Specifically, a picture is emerging from recent investigations identifying specific probiotic species to counteract the noxious effect of HM within the intestinal tract via a series of HM-resistant mechanisms. More encouragingly, aided by genetic engineering techniques, novel HM-bioremediation strategies using recombinant microorganisms have been fruitful and may provide access to promising biological medicines for HM poisoning. 

heavy metals dysbiosis probiotics molecular techniques engineered bacteria human health

1. Introduction

Rapid industrialization and urbanization have dramatically increased human exposure to heavy metals (HMs) [1], especially in developing countries. In Asia, high concentrations of HMs have been found in surface soil, drinking water and groundwater in China, Bangladesh, Vietnam, Thailand, Nepal and India [2]. In China, the total mass of HMs introduced in various waste at enormous magnitude is approximately 0.9 million tons each year [3]. Furthermore, as reported, an area of 34,000 km2 with a population of 30 million was covered by HM-polluted groundwater in six districts of India [4]. Recent accumulating epidemiological evidence suggests that high-level HM exposure causes severe damage to various organs and systems, including the kidneys, liver, the central nervous system (CNS), the reproductive system, and the hematopoietic system [5]. More than 20 types of HMs have been identified, among which cadmium (Cd), lead (Pb), and inorganic arsenic (As) were considered the most hazardous elements [6]. Based on the duration, HM exposure can be mainly classified into acute (1–14 days), intermediate (15–354 days) and chronic (≥365 days). Actually, compared to acute HM poisoning commonly induced by skin contact, the inhalation of large amounts of HM vapors, or drug misuse within a short time, chronic HM poisoning resulting from inconspicuous daily exposure through food, water, air or skin is more commonly encountered in the clinic and poses a serious threat to public health [7]. At present, chelation therapy has been the mainstay treatment for HM poisoning and related disorders; however, the use of conventional chelating agents is associated with common side effects such as adverse drug reactions, gastrointestinal distress, loss of essential metals, and strong clinical nephrotoxicity [8][9]. For this this reason, the exploration of more specific and safer therapeutic strategies for HM intoxication is the need of the hour.
The human gut microbiota, as a “superorganism” composed of 400–1000 bacterial species, fulfills many crucial roles in maintaining human health including nutrient metabolism, maintenance of mucosal integrity, immune system modulation, and protection against pathogens [10][11]. Such dynamic bacterial communities are susceptible to changes in the host conditions induced by extrinsic substances; as a result, disturbance of the gut microbiome called dysbiosis can affect the health status of the host and may end up in disease conditions [12][13]. Recently, a strong bidirectional relationship between HM exposure and gut microbiota was proposed by studies from rodent models to other advanced models [14][15][16]. It is evident that HM exposure, especially chronic exposure alters the gut microbiome trajectory and phylogenetic diversity, which may thus perturb the metabolic and physiological functions of gut microbiota, consequently, leading to the rise of many pathological conditions and toxic symptoms after HM exposure [17][18]. Reciprocally, the gut microbiota act as the primary defense against HM toxicity by affecting the intestinal HM absorption and metabolism, whilst enhancing the fecal HM excretion [19][20]. In the meantime, probiotics have received special attention due to their remarkable HM-binding capability. Generally, probiotics are termed as mono or mixed cultures of selective viable microorganisms that confer health-promoting benefits to the host by improving the balance of the intestinal micro flora [21], which traditionally include those microorganisms derived from the species of Bifidobacterium, Lactobacillus, Streptococcus, Enterococcus, Clostridium, Bacillus, and Escherichia coli (E. coli) [22]. Some classic probiotic strains, especially Lactobacillus, Bacillus, Bifidobacterium and Clostridium species have been shown to adapt strong HM resistance through alteration of the physiological conditions [23][24], or expression of HM-binding peptides/proteins [25][26] or detoxification enzymes involved in HM biotransformation [27] to reverse HM-induced dysbiosis, thereby delivering a defense against HM intoxication. However, since the health benefits of these strains appeared to be limited and ineffective, next-generation probiotics, especially bioengineered bacteria, have emerged as novel protective and therapeutic bioagents in many fields [28][29]. Great progress has been made in the application of engineered probiotics to deliver therapeutic molecules to target tissues, or express specific enzymes for local cleavage of prodrugs, or function in tandem with the host immune system to induce tolerance [30]. To date, recombinant bacteria have been used in attempts for the treatment of metabolic disorders such as diabetes [31], phenylketonuria (PKU) [32], autoimmune diseases such as inflammatory bowel disease [33], and cancer [34]. Based on this, the aid of engineered bacterial transformation technology opens up possibilities to design and develop genetically modified microbes with the desired characteristics and functionalities towards HM detoxification, which represent a promising generation to alleviate chronic HM toxicity and fill the gap in current therapeutic strategies.

2. Potential Application of Engineered Bacteria in the Detoxification of Heavy Metals

Although the majority of natural probiotics do not display infectivity or pathogenicity, the safety concerning the application of probiotic-based bioagents still remains a grey area. Contrary to expectations, some probiotic strains, particularly the enterococci were reported to pedal a string of genes related to transmissible antibiotic resistance. Bacillus cereus was also identified with the potential to produce emetic toxins and enterotoxins [35]. Moreover, it was observed that probiotic intervention caused some common side effects of deleterious metabolic activities, systemic infections, gene transfer, and excessive immune stimulation in susceptible individuals [36]. In fact, the HM-resistant properties are limited to particular microbial species, and it is necessary to combine several different strains together through oral consumption to warrant the therapeutic efficacy. It is therefore likely that complicated relationships and conflicts still exist in applying different probiotic strains, which arouse public concerns over the health risks associated with probiotic-based therapy [37].
In virtue of the serious defects in natural-origin probiotics, engineered probiotic microbes with the ideal characteristics and functionalities have emerged as an intense focus in recent years. Some strains such as Lactic acid bacteria (LAB) and E. coli Nissle 1917 (EcN), have limited therapeutic effects themselves in practice, whereas, the introduction of synthetic biology engineering is helpful to strengthen the overall effectiveness of these probiotics [38]. In detail, the survival and byproduct formation of LABs can be apparently limited in the presence of bacteriophages in the gut microbial ecosystem, for instance, the virulent Lactobacillus phage Lb338-1 was reported to exert a negative influence on the beneficial effects of the Lactobacillus strains [39]. To overwhelm this defect, specific phage-resistant bacteria have been developed by genetic modification, and further studies have shown that the mutant-engineered strain not only preserved the same probiotic features as the original parent strains, but was also equipped with antimicrobial properties against virulent pathogens [40]. On the other hand, EcN, one of the best commercially used probiotic strains in many European countries [41], has been particularly documented for the treatment of patients with ulcerative colitis (UC) [42]. The main mechanism of its action is thought to be its capability to induce the production of human β-defensin 2 (HBD2) in intestinal epithelial cells [43]. However, EcN appeared to be ineffective in the remission of Crohn’s disease (CD), due to the primary lack of defensin synthesis in the intestinal tract of CD patients [44]. Based on this, an engineered EcN strain with defensin-producing and secreting functions has been constructed, which was capable of synthesizing human α-defensin 5 (HD5) and HBD2 derivatives that might act as a delivery vehicle for these defensin-deficient patients [45].
To date, aided by the continuous development of synthetic biology and recombinant DNA technology, the therapeutic implications of genetically engineered microorganisms (GEMs) have made remarkable strides. Although it is still in the trial phase, a range of metabolic disorders, autoimmune and inflammatory diseases, infectious diseases, cognitive dysfunctions, and cancer have been targeted by the delivery of engineered probiotics and have achieved satisfactory effects of amelioration [29][46][47][48]. In principle, engineered microorganisms exert their therapeutic effects mainly through the delivery of a vaccine or drug, modulating the host immune response, imitating surface receptors or aiming at particular toxins or pathogens within the intestine to perform in situ activities [49]. Compared to traditional pharmacotherapy, engineered probiotic mediated treatments own many irreplaceable advantages such as high stability, increased standby time, reduced systemic exposure, lower delivery cost and local targeting to mucosal surfaces.
In the past decades, the application of GEMs for HM removal has attracted considerable attention due to their low cost, flexible adaptability, and eco-friendly properties. GEMs with intense degradative capacity have been widely used for the bioremediation of HMs in groundwater, soil, and activated sludge conditions. Generally, the construction of potent HM-resistant GEMs is mainly based on two strategies: (1) surface functional complexes with a high HM-binding capacity for biosorption, (2) metal ions are transported into the cytoplasm and subsequently processed by storage systems for enhanced intracellular bioaccumulation [50]. In this part, emphasis will be placed on various HM-chelating peptides/proteins, transport and storage systems and related molecular technologies that are commonly employed for the surface-adsorption and bioaccumulation of HMs.

2.1. Surface-Displayed Proteins/Peptides for Heavy Metal Biosorption

Biosorption is a metabolism-independent process by which metal ions can be adsorbed and immobilized onto the cell surface via physicochemical reactions or surface precipitation [51]. Recently, the application of molecular biology has enabled the display of foreign metal-binding proteins/peptides or other tailor-made binding proteins on the cell surface to exhibit enhanced HM biosorption. The basic principle of different bacterial display systems is fusing the heterogenous HM-binding proteins (target protein) with naturally occurring anchoring proteins (carrier protein) via three connection approaches, C-terminal fusion, N-terminal fusion, or sandwich fusion (Figure 1a), but meanwhile maintaining the independent spatial structure and biological activity of both proteins [52].
Figure 1. The procedure and principle of surface engineering towards metal adsorption in Gram-negative bacteria. (a) The coding DNA of target metal-binding peptides/proteins can be obtained from genome or plasmid DNA. After cloning it is transformed into the genome of host bacteria via fusion with the coding gene for an anchor protein (membrane protein) by one of the three different recombinant ways. Subsequently, by specific induction, the recombinant gene undergoes transcription, translation, and translocation into the cell surface. Based on different genetic recombinations, the target metal-binding peptide can be immobilized with the anchoring protein in the cell surface by C-terminal fusion, sandwich fusion or N-terminal fusion; (b) commonly used surface display systems in Gram-negative bacteria are described as follows: outer membrane proteins (OMPs): OmpA, OmpC, LamB, Ice nucleation protein (INP) and Lpp-OmpA; autotransporter: IgA protease; flagella. By these surface display systems, various metal-binding proteins/peptides can be anchored onto the outer membrane to adsorb specific metal ions. Metallothionein (MT) and phytochelatin (PC) are the most investigated metal-binding peptides that have been anchored to IgA protease [53] and OmpA [54], respectively. By Figdraw ( accessed on 3 Arpil 2022).
Notably, the surface display systems are entirely different in Gram-negative and Gram-positive bacteria owing to their distinct cell structures. Gram-negative bacteria have an extra outer membrane thus making membrane-spanning anchoring proteins necessary as a functional carrier for connection with the target external proteins. The most frequently used surface-display systems in Gram-negative bacteria contain outer-membrane proteins (OMPs), autotransporters, and the surface organelles fimbriae/flagella [52]. Among the various OMPs, ice nucleation protein (INP), Lpp-OmpA, OmpA, OmpC, LamB have been extensively applied to design engineered Gram-negative bacteria with HM adsorption capability [55]. In the autotransporter system, IgA protease β-domain is the best-known protein that has been successfully used to immobilize mouse metallothionein I (MT) protein on the cell surface [56]. Furthermore, the FimH protein, an integral part of type 1 fimbriae has been explored to display HM-binding peptides for the bioadsorption of Cd2+ and Ni2+ [57]. (Figure 1b). Whereas for Gram-positive bacteria, on account of their thick peptidoglycan layer and lack of outer membrane, the surface proteins (target proteins) are covalently linked to the peptidoglycan cell wall instead of membrane-spanning. Staphylococcal protein A (SpA) is the most-investigated anchor protein in Gram-positive bacteria [58]. Several HM-binding proteins/peptides, including two polyhistidyl peptides (HHHEHHH and HHHHHH) and screened variants of cellulose-binding domain (CBD) have been surface displayed on Gram-positive bacteria via the SpA system for HM removal [59].
In recent years, an increasing number of novel metal-binding peptides/proteins have been identified and expressed on bacterial surfaces via various display systems for HM removal. Metallothioneins (MTs) and phytochelatins (PCs) are the most classic HM-chelating proteins with desirable affinity and specificity towards Pb, Hg, As, Cd, and Ag in most genetic engineering cases [60][61]. PCs have been surface-displayed on Moraxella spp., Pseudomonas putida (P. putida)Caulobacter crescentus and Mesorhizobium huakuii that exhibited desirable biosorption for Hg and Cd [62]. Surface engineering of MTs on Ralstonia eutropha and E. coli cells indicated a 15–20 times higher Cd accumulation compared to the wild-type strains [61][63]. Apart from MTs and PCs, a surprising array of novel metal-binding domains have been explored for their potential application in HM removal. For instance, three copper-binding peptides NRWHHLE, NAKHHPR, and SPHHGGW [64], two Cd-binding peptides polyhistidine His12 [65] and SynHMB [66], three Pb-binding proteins PbrR, PbrR691, and PbrD [67] were surface-expressed in different strains to adsorb HMs from the contaminated sites.

2.2. Transport and Storage Systems for Heavy Metal Bioaccumulation

Bioaccumulation is another probiotic-adapted mechanism to tolerate HM stress. It is a metabolically active process responsible for transporting HMs into intracellular space and subsequently secluding them by metal-chelating proteins/peptides or biotransforming them via enzymatic reactions. The most well-known import systems for HMs include three major transporter classes: primary active transporters, channels, and secondary carriers [68] (Figure 2). In detail, primary active transporters spontaneously carry HMs into cells against a concentration gradient by hydrolysis of ATP/GTP. Secondary carriers are capable of translocating cationic HMs into cytoplasm driven by the proton-motive force (PMF). Channels are energy-independent transporters that engage in the passive diffusion of HMs into the cells along their concentration gradient [69]. In recent years, vast experimental attempts have explored the utilization of several ion import systems to strengthen HM uptake by genetic engineering. Primary active transporters MntA and cdtB from L. plantarum and TcHMA3 from the flowering plant Thlaspi caerulescens have been recombinantly expressed in engineered strains for the efficient import of Cd [70][71]. Secondary carriers NixA and its homologs have been used to improve Ni and Co uptake, and Hxt7, Pho84 from Saccharomyces cerevisiae have been identified for As removal [72][73]. Moreover, researchers have discovered channels of homotetramer glycerol facilitators (GlpF) [74] and homolog Fps1 [75] with a specific affinity toward As, and MerT/P, MerC, MerE, and MerF [76] for the transport of Hg in the bioaccumulation process.
Figure 2. Import system and storage system adapted in Gram-negative bacteria towards bioaccumulation. Biosorption is indicated in the left side. The import system used in bioaccumulation includes primary active transporters (requiring NTPs such as ATP), secondary carriers (requiring a proton concentration gradient), and channels (no energy needed), by which metal ions are imported across the inner lipid membrane into the cytoplasm and undergo the process of the storage system. The storage system is responsible for HM sequestration by attachment to different metal-binding proteins/peptides (represented by PCs and MTs) or HM biotransformation by various detoxifying enzymes (represented by Hg reductase and As methyltransferase). By Figdraw ( accessed on 1 April 2022).
Immediately after the import of HMs into the intracellular space, multiple storage systems serve as the key to sequestrating or detoxifying HMs for the release of the HM-stimulated oxidative stress and inflammatory responses. In most cases, the HM-storage systems refer to genetically encoded metal-binding proteins/polymers (MBPs), or a catalogue of enzymes that biotransform metal ions into less toxic forms. In addition to the best-known metal-binding proteins PCs and MTs, Hpn (UniProt P0A0V6) from Helicobacter Pylori (H. pylori) [77], and (UniProt Q9FCE4) from Streptomyces coelicolor [78] have been identified to bind Ni in the process of bioaccumulation. On the other hand, A Hg resistance gene (mer) operon coding for a Hg reductase that converts Hg2+ into volatile Hg is an elegant example of enzyme-mediated HM storage systems. More tellingly, this Hg resistant system aids in the Hg2+ import followed by enzymatic reduction of toxic Hg2+ into non-toxic Hg0, and ultimately leads to Hg0 diffusion outwards from the cells [79]. Several recombinant strains, including Acidithiobacillus ferrooxidans with over-expressed merC gene, E. coli with transferred merT–merP genes, and D. radiodurans with cloned merA gene have shown an ideal capability to detoxify Hg2+ [80][81]. In addition, arsM gene encoding S-adenosylmethionine methyltransferase for the conversion of toxic inorganic As to less toxic volatile trimethylarsine (TMA) has been obtained from Rhodopseudomonas palustris [82]. Overexpression of the arsM gene in Sphingomonas desiccabilis and Bacillus idriensis have exhibited promising As detoxification [83].

2.3. In Vivo Attempts for Heavy Metal Detoxification by Engineered Strains

In attribution to the successful application of GEMs in the field of environmental HM bioremediation, biomedical workers were inspired to explore the feasibility of developing engineered bacteria as biological medicines for HM poisoning. In this aspect, surface display techniques and various HM transport/storage systems might be effective tools to design tailor-made GEMs with intense detoxification capabilities, which may be directly inoculated into the intestine by oral consumption, thereby counteracting the toxic effects of HMs within the intestine. However, up to now, only very few engineered bacteria have been developed or applied in vivo as a biological treatment against HM poisoning. After the present probe into pre-clinical research work, the following is a brief description of three currently available pieces of research on animal models that relate to this topic.
In 2018, Chan and his coworkers developed a novel whole-cell biosorbent for Pb by displaying PbrR, a well-investigated protein with Pb-binding capacity, on the cell surface of E. coli using Lpp-OmpA as the anchoring motif. In vitro, the results of atomic absorption spectroscopy (AAS) suggested that the PbrR-displayed cells had a four-times higher binding of Pb than non-PbrR cells, effective at both neutral and acidic pH. In vivo, a pre-experiment revealed that no noxious effects or pathogenic potential were observed after oral administration of PbrR-displayed cells on male Kunming (KM) mice. In order to determine the Pb-binding capacity of the PbrR-displayed E. coli, Pb2+ was supplied in the forms of lead acetate, saturated lead in PbrR-displayed E. coli, and unsaturated lead in PbrR-displayed E. coli to male KM mice at a dose of about 20 μg Pb/mouse daily for more than 15 days (human maximum Pb tolerable intake: 3 µg per day for children and 12.5 µg per day for adults). Encouragingly, the Pb concentration deposited in murine blood and bone was significantly reduced in all mouse groups treated with PbrR-displayed E. coli. compared to the Pb-only group. Furthermore, the metabolization of essential metals (Ca, Mg, Fe, Zn, and Cu) was not affected by the oral administration of PbrR-displayed E. coli [84].
In 2019, the team of Minrui Liu constructed two surface-displayed E. coli strains for selective adsorption of Hg2+ and its derivative MeHg, respectively [85][86]. For Hg2+ removal, a novel Hg2+-binding peptide encoded by the sequence of CysLysCysLysCysLysCys (CL) was surface-expressed on the E. coli BL21 by anchoring to the N-terminal region of the ice nucleation protein (INP-N). Similar to the last report, the in vitro AAS tests indicated a four-fold higher Hg2+ adsorption in CL-displayed E. coli than the control strain. Furthermore, the CL-displayed E. coli exhibited a strong specificity toward Hg2+ in the co-existence of other metal ions (Cd2+, Pb2+, Ni2+, Zn2+ and Cu2+). In vivo, Carassius auratus (C. auratus) were fed with engineered E. coli added to the fish food at a dose of 2 × 108 CFU/g for 10 days (stage 1), and then were given 0.1 mg/kg Hg2+ exposure for 30 days (stage 2). It was shown that the Hg accumulation in fish muscles was reduced by 51.1% and the amount of Hg excretion in fish feces increased by 56.5% in the engineered E. coli-treated group compared with the non-fed group. Interestingly, it was observed that the Hg-induced increase in the relative amount of Vibrio spp. was reduced by 8.02% and the decrease in Cetobacterium was reversed by 12.00% after the oral administration of CL-displayed E. coli [85].
In the second study, engineered E. coli W-1 with surface-displayed CL peptide was constructed by the same method used in the last study. The results of the in vitro test showed that the CL-displayed E. coli W-1 exhibited a maximum MeHg adsorption rate of 96.3 ± 0.8%, whereas that of the original strain W-1 was less than 30%. Furthermore, the CL-displayed E. coli W-1 had a specific affinity towards MeHg when other metal ions (Cr3+, Zn2+, Cd2+, Ni2+, Cu2+, and Pb2+) persisted in the condition. The design of the in vivo experiment was similar to that of the study mentioned above, in brief, a 10-day inoculation of the recombinant E. coli at a level of 1 × 109 CFU/g followed by a 30-day MeHg feed at a dose of 0.05 mg/kg on C. auratus. After DNA extraction from the intestinal contents of each individual fish, qPCR was used to detect the colonization and persistence of the engineered E. coli W-1 in fish intestines. The results indicated that, compared to the non-detectable proportion of engineered E. coli W-1 in the gut of the fishes from the control group, in the groups fed with engineered strains, the proportion of engineered E. coli W-1 accounted for 0.5% of the total bacterial community in the fish guts at the end of the in vivo experiment. This revealed that the engineered bacteria successfully colonized in the C. auratus intestine and persisted for an adequate period until the end of the 30-day MeHg feed, thus giving rise to the following result concerning MeHg reduction in fish tissues. As expected, compared to the untreated group, the accumulated MeHg concentrations in fish tissue were decreased by 36.3 ± 0.7% and the amount of MeHg excretion in feces was increased by 36.7 ± 0.8% in the groups treated with CL-displayed E. coli W-1 [86].
Taken together, the data reported here both support the idea that oral administration of surface-engineered bacteria is an effective approach to adsorb HMs in the intestine, thus reducing HM accumulation in the body and protecting animals from HM-induced toxicity. Collectively, the following findings should be highlighted from the present studies, which may provide new ideas for the researchers to understand how GEMs aid in the intra-intestinal detoxification of HMs. Firstly, it should be noted that Lpp-OmpA and INP, the anchoring systems previously used for in vitro bioremediation, were also effective in immobilizing PbrR and CL proteins on the cell surface for in vivo application. Secondly, the evidence from these studies demonstrates that the recombinant bacteria could persist and vitally function for HM biosorption at different PHs, even in the extreme acidic conditions simulating the stomach. For instance, the amount of adsorbed Pb by PbrR-displayed E. coli was still 6.2-fold higher than undisplayed E. coli at pH 3.0 [84]. Thirdly, in all three studies, no noxious effects on animal growth nor intestinal epithelial invasion were observed after oral administration of engineered strains, which provides an experimental basis for safety assessment concerning their in vivo application. Further, the common property of the three engineered bacteria refers to a selective affinity towards the target HM even in the co-existence of other metals. This feature highlights one of their advantages, that is, the blood levels of physiologically essential metals will not be affected by their oral administration, overwhelming the conventional chelating agents with a side effect of essential metal loss. Last but not least, another noteworthy finding is that the oral administration of engineered E. coli W-1 reversed the HM-induced compositional alterations of gut microbiota [87]. A possible explanation for this might be that the host strain E. coli W-1, a commensal bacteria isolated from the fish intestine, has an innate probiotic property to counteract the HM toxicity by rebuilding the microecology. It can therefore be assumed that if probiotic species can be chosen for genetic engineering, their inherent beneficial properties will strengthen the detoxification effect of engineered bacteria and accelerate the process of intra-intestinal removal of HMs. In conclusion, the combination of these findings, at least preliminarily, suggests that the surface-display technologies conferred commensal bacteria- or probiotic-enhanced capability for in vivo HM adsorption, which lays the groundwork for future research into GEM-based preventive or therapeutic bioagents for HM intoxication. Finally, the most important source of weakness in the current studies needs to be illustrated here. The three in vivo experiments were not based on the model of chronic HM exposure, so fails to specify whether GEMs have the potential to be orally supplied for the treatment of chronic HM intoxication.


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