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Toxicity of Ionic Liquids
Chapter 13
Toxicity of Ionic Liquids: Past, Present, and Future Diego O. Hartmann, Cristina Silva Pereira Universidade Nova de Lisboa, Instituto de Tecnologia Quı´mica e Biolo´gica Anto´nio Xavier, Oeiras, Portugal
Ionic liquids (ILs) have been described as molten salts that have, by definition, a melting point below 100 C (Stark and Seddon, 2007). They are entirely ionic in nature and usually consist of a large asymmetrical organic cation associated with a polyatomic anion that may be either organic or inorganic. In the design of ILs, the asymmetry of the cation or anion is preferred over symmetrical species, since the former do not tend to pack so easily, therefore rendering salts with a lower melting point (Stark and Seddon, 2007). Structural modifications can be made either to the anion, to the cation, or in substituents on these two species. Therefore, millions of formulations become possible and, by altering their cationic or anionic components, their properties can be tailored (Plechkova and Seddon, 2007; Stark and Seddon, 2007). Their physical and chemical properties (eg, melting point, viscosity, solubility, or hydrophobicity and chemical polarity or hydrogen bonding ability, respectively) can be tuned to best suit a specific process. The tunability of such properties rendered ILs an excellent choice as alternative solvents in many chemical processes. They already found applications in many reactions (Plechkova and Seddon, 2008), as solvents (Holbrey and Seddon, 1999; Plechkova and Seddon, 2008) and catalysts (Cole et al., 2002), and show great potential in electrochemistry (Armand et al., 2009; Sakaebe et al., 2007). ILs’ physical and chemical properties, together with their negligible vapor pressure and bulk nonflammability, suggested them as potential green solvents (Deetlefs and Seddon, 2006, 2010; Dong et al., 2007). Their negligible volatility distinguishes them from traditional organic solvents, reducing risk of atmospheric pollution. However, the potential release of IL vapors should be considered when used at elevated temperatures (Ranke et al., 2007a,b). Nonetheless, soil and water contamination, either by accidental spills or waste disposal, constitutes a serious hazard. The ecotoxicological assessment of this class of compounds should be considered a priority, and has been taken into Ionic Liquids in Lipid Processing and Analysis. http://dx.doi.org/10.1016/B978-1-63067-047-4.00013-1 Copyright © 2016 AOCS Press. Published by Elsevier Inc. All rights reserved.
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account for the conscious design of novel ILs (Petkovic et al., 2011; Petkovic and Silva Pereira, 2012).
13.1 PAST: EXTENSIVE STUDIES ON IONIC LIQUID TOXICITY Interest in IL toxicity began in the early 2000s. Pernak et al. (2001a,b) developed new cationic surfactants with benzimidazolium or pyridinium cores and evaluated their antimicrobial properties (minimal inhibitory and lethal concentrations against bacterial and yeast strains relevant for human health). The evaluation of the antimicrobial activity of 1-alkoxymethyl-(3-nicotionylaminomethyl)benzimidazolium, 1-alkoxymethyl-3-(1-benzimidazolmethylamino)pyridinium, 1-alkoxymethyl-3-[1-(benzotriazol-1-yl)methylamino]pyridinium chlorides, and N,N0 -bis[3-(1-alkoxymethyl)pyridinium chloride]methylenediamine revealed that their toxicity increased with the elongation of the alkoxy chain, which varied between 2 and 12 carbon atoms. This constituted one of the first systematic studies on the toxicity of ILs, and served afterward as a basis for evaluating the toxicity of many newly synthesized imidazolium- and pyridinium-based compounds (Fig. 13.1), including 1-alkoxymethylcarbamoylpyridinium chlorides (Pernak et al., 2001a,b), 1-alkyl-3-methylimidazolium chlorides, and bromides; 1-alkyl-3-hydroxyethyl2-methylimidazolium chlorides (Demberelnyamba et al., 2004), 1-alkoxymethyl-3-methylimidazolium chlorides, tetrafluoroborates and hexafluorophosphates (Pernak et al., 2003), 1,3-dialkoxymethylimidazolium chlorides (Pernak et al., 2004a,b,c), 1-alkyl- and 1-alkoxy-methylimidazolium lactates (Pernak et al., 2004a,b,c), 1-alkoxymethyl-3-hydroxypyridinium, and 1-alkoxymethyl-3-dimethylaminopyridinium chlorides (Pernak and Branicka, 2003). The data revealed a clear trend toward a stronger toxic effect of ILs with the increase in the length of the alkyl or the alkoxy side chain. Further studies revealed that the incorporation of an oxygen atom in the side chain significantly reduced the toxicity of the ILs (Morrissey et al., 2009). Symmetrical chains in positions R1 and R3 of the imidazolium ring, however, resulted in lower toxicity, probably due to steric effects with the cell surface (Pernak et al., 2004a,b,c). The effect of anions on the observed toxicities seemed to be secondary to the effect of the cations, but their broad diversity did not allow a conclusive analysis. These initial observations inspired an escalating number of studies on toxicity of several ILs, expanding the research for other biological models, particularly model eukaryotic organisms. For example, the toxicity of ILs with 1-alkyl-3-methylimidazolium as cation were analyzed against leukemia and glioma rat cell lines (Ranke et al., 2004), human HeLa cell lines (Stepnowski et al., 2004), and the worm Caenorhabditis elegans (Swatloski et al., 2004). Additionally, the toxicity of pyridinium-based ILs has been analyzed using freshwater snail Physa acuta (Bernot et al., 2005), the zebra mussel Dreissena polymorpha (Costello et al., 2009), and green algae Pseudokirchneriella subcapitata (Pham et al., 2008), focusing on the effect of the alkyl side chain
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FIGURE 13.1 Common IL cation cores and some of their possible structural modifications: (A) imidazolium, (B) pyridinium, (C) quinilinium, (D) pyrrolidinium, (E) piperidinium, (F) morpholinium, (G) quaternary ammonium, and (H) quaternary phosphonium cations.
of the pyridinium cation. The effects of imidazolium- and pyridinium-based ILs in the bioluminescence of the bacterium Vibrio fischeri were also analyzed (Docherty and Kulpa, 2005). Bacterial bioluminescence is directly linked to cellular respiration, so decrease in luminescence is indicative of toxicity. Other cationic head groups were also evaluated on their toxic effects, such as the aromatic quinolinium-based cations (Fig. 13.1). Despite rarely being studied, these ILs showed great antimicrobial properties and cytotoxicity potential that increased with elongation of substituted alkyl chain (Busetti et al., 2010; Ranke et al., 2007a,b). The effects of ILs with nitrogen-containing alicyclic cations, namely pyrrolidinium, piperidinium, and morpholinium (Fig. 13.1), were also investigated; for example, their aquatic toxicity against the zebrafish Danio rerio (Pretti et al., 2006) and their cytotoxicity against mammal cell lines (Kumar et al., 2009; Stolte et al., 2007a,b). It was observed that increasing numbers of carbon atoms in an alicyclic ring generally increases toxicity; for example, the piperidinium cation (six-member rings) was more toxic than the pyrrolidinium cation (five-member rings) (Kumar et al., 2009). The morpholinium ILs, due to incorporation of an
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oxygen atom in the ring, were the least toxic of these alicyclic cations. Furthermore, the nonaromatic head groups appeared to be generally less toxic than their aromatic equivalents (Kumar et al., 2009; Stolte et al., 2007a,b). Their lower toxicity, relative to the corresponding aromatic rings, was generally evident in the bioassays with models such as V. fischeri, the algae Scenedesmus vacuolatus, and the aquatic plant Lemna minor (Stolte et al., 2007a,b). As observed for imidazolium- and pyridinium-based ILs, the length of the alkyl chains in the cation played a major role in toxicity of piperidinium- and pyrrolidinium-based ILs (Kumar et al., 2009; Ranke et al., 2007a,b). Anions have been considered to have an unpredictable effect, largely reflecting the lack of systematization in their ecotoxicological assessment. Quaternary ammonium salts (Fig. 13.1) have been largely studied and explored in numerous applications, such as disinfectants, surfactants, antistatic agents, and catalysts (Jones, 2001). Their properties depend on chain length and functional groups of anion (Ying, 2006). Pernak and coauthors were the first to consider their antimicrobial activity against relevant clinical strains, which was, as expected, governed by length of the alkoxy and alkyl side chains (Pernak and Chwała, 2003; Pernak and Feder-Kubis, 2005). The toxicity of ammonium ILs against other biological models was also tested, including bioluminescent bacterium V. fischeri (Couling et al., 2006), fresh water crustacean Daphnia magna, and algae P. subcapitata (Pretti et al., 2009; Wells and Coombe, 2006), zebrafish (Pretti et al., 2009), and cell lines (Ranke et al., 2007a,b). The same toxicity trends previously described for other cationic head groups were observed; the highest toxicity was detected for those with longest alkyl chains and could be well correlated with the cation lipophilicity. Some of the most interesting groups of quaternary ammonium-based ILs are those containing the cholinium cation (2-hydroxyethyl-trimethylammonium). Its combination with benign anions constituted a major advance in the design of biocompatible ILs (Abbott et al., 2001, 2002, 2003, 2004; Abbott and Davies, 2000; Fukaya et al., 2007; Pernak et al., 2007; Renshaw, 1909). The low toxicity of many cholinium ILs has been demonstrated, including those carrying as anions; for example, saccharinate and acesulfamate (Nockemann et al., 2007), dimethylphosphate (Dipeolu et al., 2009), phosphate-based anions (Weaver et al., 2010), lactates (Petkovic et al., 2009), and alkanoates (Petkovic et al., 2010). The last were analyzed by our group for the first time against filamentous fungi of the genus Penicillum, and included a range of linear alkanoate anions and two structural isomers (Petkovic et al., 2010). Their toxicity increased with the elongation of the anion, with the branched isomers less toxic than corresponding linear ones with an equal number of carbon atoms. These cholinium alkanoates also displayed high biodegradability potential and great solvent ability (Garcia et al., 2010). These findings have been strengthened by other groups, which also evaluated their cytotoxicity (Klein et al., 2013). The research focus on toxicity of ILs is moving toward other cationic groups aside imidazolium- and pyridinium-based ones, such as the quaternary
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phosphonium cations (Fig. 13.1). These ILs are currently produced in tonne quantities, particularly by Cytec Industries Inc. (Bradaric et al., 2003), further highlighting the need for their ecotoxicological assessment, which is still seldom investigated. Some halides (eg, with the tetrabutylphosphonium cation) showed levels of toxicity against the freshwater snail P. acuta that were comparable to imidazolium-based ILs carrying the same chain length (Bernot et al., 2005). The antimicrobial properties of a series of alkyltrihexylphosphonium halides demonstrated a significant role of the structure of the cation in their toxicity, since the antimicrobial activity decreased for the longest alkyl chains (8e14 carbon atoms) (Cieniecka-Rosłonkiewicz et al., 2005). The apparently high toxicity of tetraalkylphosphonium halides against V. fischeri, and D. magna (Couling et al., 2006; Wells and Coombe, 2006), and P. subcapitata (Cho et al., 2008; Wells and Coombe, 2006) was also demonstrated, as well as their cytotoxicity against human and rat cell lines (Ranke et al., 2007a,b, Wang et al., 2007). The lack of systematization in these studies, however, did not allow a conclusive rationalization, a general problem when evaluating the toxicity of the highly diverse groups of ILs (Petkovic et al., 2011; Petkovic and Silva Pereira, 2012). These initial studies were essential to establish the foundations for evaluating the ecotoxicity of ILs, particularly for understanding how the cationic or anionic head groups and their structural modifications impact toxicity. Despite the massive amount of significant data produced so far, evidence of their specific mechanisms of toxicity is still largely lacking. In-depth knowledge of their toxicity is critical for developing safer IL based-processes and may provide unforeseen opportunities in IL frontier research.
13.2 PRESENT: UNDERSTANDING THE MOLECULAR BASIS OF IONIC LIQUID TOXICITY The hydrophobic interactions between ILs and biological membranes have been proposed as a main mechanism of toxicity of ILs, largely supported by the observation that their toxic effects increase with the elongation of the side chains. This nonspecific toxicity, called baseline toxicity or narcosis (Mackay et al., 2009), appears to be correlated with 1-octanol/water partition coefficients. This is suggestive of an increase in either the cation or anion lipophilicity with elongation of the side chains (Stolte et al., 2007a,b), leading to greater probability of interaction with biological membranes. Liposomes (ie, phospholipid vesicles) were used to assess the effects of some ILs in cellular membranes (Schaffran et al., 2009). As an example, the 1-octyl-3-methylimidazolium cation was demonstrated to cause severe disruptions in a supported phospholipid bilayer (Evans, 2008). Molecular dynamics simulation studies also tried to demonstrate the interactions of ILs with artificial lipid bilayers (Klein et al., 2013; Ruegg et al., 2014). These elegant models are, however, unable to reproduce the complexity of
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the heterogeneous lipid bilayers of biological membranes. Therefore, real biological systems, with all their intrinsic complexity, are required to evaluate the true effects of ILs. The lack of systematic studies on membrane permeabilization as a mechanism for toxicity of ILs was the driving force of our work. We pioneered the use of filamentous fungi ecotoxicological assessment of ILs (Petkovic et al., 2009). They were shown to tolerate, in general, very high concentrations of ILs. Moreover, their high catabolic capacities render them as great candidates for biodegradation studies. We have successfully established all the methodologies necessary to evaluate the effects of ILs against filamentous fungi, excellent model organisms for moving beyond the state-of-the-art on ILs toxicity. The first ILs subjected to in-depth investigation of their mechanisms of toxicity was a series of highly toxic alkyltributylphosphonium chlorides, [P4 4 4 n]Cl, where n ¼ 1, 4e8, 10, 12, or 14. A fluorescence microscopy assessment demonstrated that these ILs were able to permeabilize the plasma membrane of the spores of the model filamentous fungus Aspergillus nidulans (Fig. 13.2) (Petkovic et al., 2012). Their toxicity was observed to increase with systematic elongation of one alkyl substituent, consistent with the increase in their 1-octanol/water partition coefficient. The data clearly suggested that the toxicity of [P4 4 4 n]Cl, where n 4, is ruled by direct interaction with the plasma membrane. Further confirmation of these effects was attained by analyzing IL effects at a molecular level, in particular by measuring the expression levels of genes involved in plasma membrane biosynthesis in A. nidulans (Hartmann and Silva Pereira, 2013). The data suggested that the fungus alters the plasma membrane fluidity in response to the membrane permeabilization provoked by the ILs. Both fluorescence microscopy and gene expression analysis were proven to be powerful and rapid tools to investigate in vivo the ability of ILs to permeabilize biological membranes. Accordingly, these methods were used to investigate the toxic effects of other important families of ILs. The imidazolium-based ILs are probably the most investigated group and one of the first to find application on an industrial scale (Plechkova and Seddon, 2008). The cholinium-based ILs are currently gaining great interest for the development of biocompatible ILs, mainly due to the benign nature of the cholinium cation (Klein et al., 2013; Pernak and Chwała, 2003; Pernak et al., 2007; Petkovic et al., 2010). We have chosen representative families of each of these IL groups, namely 1-alkyl-3-methylimidazolium chlorides ([Cnmim]Cl, where n ¼ 2, 4, 6, 8, or 10) and cholinium alkanoates (anion ¼ ethanoate, butanoate, hexanoate, octanoate, or decanoate), to perform a systematic investigation of their toxic effects and mechanisms of toxicity. As previously observed for the phosphonium-based ILs, both 1-alkyl-3-methylimidazolium chlorides and cholinium alkanoates present the same trend of toxicity against A. nidulans: their toxicity increased with the elongation of the alkyl
FIGURE 13.2 Fluorescence microscopy of asexual spores of Aspergillus nidulans exposed to alkyltributylphosphonium chlorides ([P4 4 4 n]Cl, n ¼ 1, 4, 8, or 12) for 1 h and stained with propidium iodide, which only stains cells with permeabilized membranes. The left column shows the total number of spores and the right column shows membrane-damaged spores. The percentage of damaged cells increases with the elongation of the alkyl chain in the cation. Scale bar: 20 mm. Adapted from Petkovic, M., Hartmann, D.O., Adamova´, G., Seddon, K.R., Rebelo, L.P.N., Silva Pereira, C., 2012. Unravelling the mechanism of toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans conidia. New J. Chem. 36 (1), 56e63.
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chain either in the cation or the anion, respectively. However, while imidazolium-based ILs appear to be toxic and recalcitrant to degradation, cholinium alkanoates present low toxicity and are readily biodegradable (Petkovic et al., 2010). Furthermore, 1-alkyl-3-methylimidazolium chlorides with long alkyl chains ([Cnmim]Cl, where n 6) were able to permeabilize the fungal plasma membrane, leading to cell death, while cholinium alkanoates could not cause such an effect (Hartmann et al., 2015). These differences were most likely due to the nature of each IL family. The plasma membrane is charged along its surface, due to the different composition of neutral and negatively charged head groups of its composing phospholipids (Alberts et al., 2002). Toxicity of 1-alkyl-3-methylimidazolium chlorides seems to closely relate to the elongation of the alkyl chain in the cation, while for cholinium alkanoates, increasing number of carbons in the anion appears to rule their toxicity. Most probably, the negatively charged phospholipids assist the interaction of the imidazolium-based cations with the plasma membrane, allowing its permeabilization, while the anions from cholinium alkanoates are likely repelled. The cholinium cation, on the other hand, can interact with the surface of the plasma membrane, but its bulky nature does not allow membrane permeabilization. Elongating one of the alkyl chains of the cholinium cation, however, enables plasma membrane permeabilization. This could be observed for a series of alkyl-(2-hydroxyethyl)-dimethylammonium bromides ([N1 1 n 2OH]Br, where n ¼ 2, 4, 6, 8, 10 or 12) (Hartmann et al., 2015). The analyses of the toxicity of these cholinium derivatives reveal that this family of ILs presented membrane-permeabilizing effects but maintains the low toxicity and the biodegradability of the archetype cholinium-based ILs. These observations constitute a unique asset for the development of new biocompatible ILs: linear elongation of alkyl chains should be preferentially introduced in the anions, in order to avoid permeabilization of biological membranes. Interaction with the plasma membrane and its disruption is most likely a nonspecific mechanism of toxicity of ILs, probably observed for all cellular organisms. In addition to the plasma membrane the fungal cell envelope comprises another complex structure, the cell wall. The cell wall of A. nidulans presents an inner layer composed of polysaccharides, mainly chitin, 1,3-b-glucans, and 1,3-a-glucan (de Groot et al., 2009). The glucan network serves as a support for an outer layer of mannoproteins. The presence of these mannoproteins confers an overall negative charge to the cell wall that might also have an implication on the effects of ILs in fungi. Employing fluorescence microscopy, gene expression analysis, and scanning electron microscopy, we analyzed the ability of ILs to cause damage to the fungal cell wall and observed that alkyltributylphosphonium chlorides, in fact, damage this cellular structure, regardless of the alkyl chain length (Fig. 13.3) (Hartmann and Silva Pereira, 2013; Petkovic et al., 2012). Other ILs with long
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FIGURE 13.3 Scanning electron microscopy analysis of asexual spores of Aspergillus nidulans exposed to alkyltributylphosphonium chlorides ([P4 4 4 n]Cl, n ¼ 1, 8, or 12) for 2 h. Images reveal that ILs cause severe damage to the cell wall and surface of the cells. Micrographs on the left and on the right have 5000 and 15,000 magnification, respectively. Scale bars: 1 mm. Adapted from Petkovic, M., Hartmann, D.O., Adamova´, G., Seddon, K.R., Rebelo, L.P.N., Silva Pereira, C., 2012. Unravelling the mechanism of toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans conidia. New J. Chem. 36 (1), 56e63.
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alkyl chains in the cation, namely 1-alkyl-3-methylimidazolium chlorides and alkyl-(2-hydroxyethyl)-dimethylammonium bromides, also induce cell wall damage (unpublished data). Cholinium alkanoates, however, do not cause such effects. It seems possible that the negative charge of the cell wall also played a pivotal role in the effects of ILs upon this cellular structure. Additional studies were undertaken by our group to further investigate the mechanisms used by filamentous fungi to tolerate ILs. We analyzed the mycelial proteome of two model fungi upon exposure to 1-ethyl-3methylimidazolium chloride and cholinium chloride, which were selected since they present opposite toxicity and recalcitrance toward fungi (Martins et al., 2013). Data obtained showed that numerous stress-responsive proteins (eg, anti-ROS defense proteins and drug transporter proteins) as well as several critical biological processes and/or pathways were affected by either IL. Among other changes, these compounds altered developmental program in both fungi, for example, promoting the formation of Hu¨lle cells (involved in sexual development of the fungus) or asexual sporulation, and led to accumulation of osmolytes, namely glycerol in Neurospora crassa and betaine in A. nidulans. This is consistent with the observation that addition of betaine to the bacterium Enterobacter lignolyticus growth media supplemented with 0.375 M of 1-ethyl-3-methylimidazolium chloride increased biomass production (Khudyakov et al., 2012). Interestingly, the proteomic study also shed light on the mechanism of toxicity of cholinium-based ILs. Major uptake of the cholinium cation led to the production of the toxic compound cyanide, which might be a cause of the observed growth inhibition provoked by this group of ILs (Martins et al., 2013). Another remarkable finding was the accumulation of a protein likely involved in the biosynthesis of unusual amino acids in N. crassa (unpublished data). In some fungi, these rare amino acids are involved in the formation of unusual metabolites, such as neoefrapeptins and acretocins, which usually show broad antimicrobial activity (Bruckner and Theis, 2010; Fredenhagen et al., 2006). Further tests are ongoing to confirm the presence of metabolites potentially carrying interesting biological activity. These results clearly demonstrate the importance of unraveling the mechanisms of toxicity of ILs at a molecular level, revealing that their full potential remains largely overlooked.
13.3 FUTURE: MOVING BEYOND TOXICITY TOWARD NEW POTENTIAL APPLICATIONS From the knowledge obtained by the ecotoxicological assessment of ILs and their mechanisms of toxicity, two paths are possible. The first is the development of new green, safer ILs and their application in environmentally friendly processes. As an example, the biocompatible and biodegradable cholinium alkanoates developed by our group (Petkovic et al., 2010) have been observed to have a great solvent ability toward the plant polymer suberin
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(Ferreira et al., 2012; Garcia et al., 2010; Petkovic et al., 2010). Cholinium hexanoate was successfully employed in the extraction of suberin from cork where it plays the dual roles of solvent and mild catalyst (Ferreira et al., 2014). This ensured the preservation of the native structure of the polymer, which allowed, for the first time, the formation of suberin films with antibacterial properties (Garcia et al., 2014). Moreover, deep knowledge on IL toxicity inspired the development of engineered bacteria carrying phenotypes with increased resistance to particular ILs in order to promote their efficient use in biorefineries and biofuel production (Khudyakov et al., 2012; Ruegg et al., 2014). The second possible path is to explore ILs in biological applications, in which their unique toxic properties become useful. A good example of their early applications was the innovative use of 1-alkoxymethyl-3methylimidazolium tetrafluoroborate in embalming and tissue preservation (Majewski et al., 2003), exploring their antimicrobial activity as a replacement for the highly volatile and toxic formalin, and as a wood preservative (Pernak et al., 2004a,b,c). The potential use of ILs as drugs for important etiological agents is also under current investigation. For example, imidazolium-based ILs have been explored for their potential in combating Plasmodium falciparum (Vlahakis et al., 2010) and Trypanosoma cruzi (Faral-Tello et al., 2014), the causative agents of malaria and Chagas disease, respectively. However, their cytotoxicity still hampers their clinical usage. A better example is choline-geranate that has been proposed as a coadjutant in the treatment of wound infections, due to its antimicrobial activity, minimal toxicity to epithelial cells as well as skin, and effective permeation enhancement for drug delivery (Zakrewsky et al., 2014). Probably one of the most interesting and promising applications of ILs is their combination with active pharmaceutical ingredients (APIs) (Marrucho et al., 2014). These pure liquid salt forms of active pharmaceutical ingredients (API-ILs) are considered a novel strategy to overcome existing problems with solid-state APIs. This strategy potentially allows the liquefaction of the drug and when combined with adequate ionic counterparts can enhance its delivery. Several drugs have already been successfully synthesized in their API-IL form, such as the antiinflammatory ibuprofen, the antibiotic amoxicillin, and the anesthetic procaine (Marrucho et al., 2014). Progress in this field requires full compliance of the new API-ILs with the extant legislation. ILs have also been explored to increase the solubility of insoluble drugs and enhance their topical and transdermal delivery (Dobler et al., 2013; Shamshina et al., 2013). Our group has ongoing side projects that aim at using IL formulations to tune the solubility of well-established cytotoxic antifungal drugs, while establishing proof-of-concept that their mechanism of activity remains unaltered. Rather unexplored is the potential of ILs to induce metabolic alterations and stress responses in living organisms that might be of great interest in biological sciences. Our studies on the mechanisms of toxicity of ILs revealed
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that some ILs not only cause plasma membrane permeabilization and cell wall damage, but also seem to induce alteration in lipid metabolism in filamentous fungi, more specifically in the synthesis of sphingolipids (unpublished data). Fungi respond to damage to the cell wall by activating several genes involved in its biosynthesis, creating conditions that allow them to reestablish its integrity. This response is regulated by a signaling cascade better understood in the yeast Saccharomyces cerevisiae, called cell wall integrity pathway (Levin, 2005). This pathway is also known to be present in a variety of filamentous fungi, but many knowledge gaps in their constitution and regulation call for further investigation. It is thought that A. nidulans possesses an alternative pathway for cell wall integrity regulation, still uncharacterized (Fujioka et al., 2007). Exposure to alkyltributylphosphonium chlorides, as an example, led to cell wall damage and was believed to induce the activation of an alternative cell wall integrity pathway (Hartmann and Silva Pereira, 2013). This scientific hypothesis is tremendously exciting, especially when considering the significance of this pathway as a potential pathogen-specific target for the development of new antifungal drugs. Importantly, there are only three major classes of antifungal drugs in clinical use and the current pace of drug development is inadequate to fight the threat of emerging fungal pathogens and multidrug resistance (Ostrosky-Zeichner et al., 2010). Interfaces of the cell wall integrity pathway with other signaling pathways in S. cerevisiae have been observed, including the sphingolipid metabolism (Levin, 2005). This, however, has not yet been elucidated in the filamentous fungus A. nidulans. We have observed that some ILs that cause membrane permeabilization and cell wall damage also activate the sphingolipid biosynthetic pathway and induce accumulation of certain sphingolipid intermediates, such as sphingoid bases (unpublished data). It is possible that these molecules are acting as second messengers for the response to cell wall stress induced by ILs. Curiously, each family of ILs tested seemed to induce distinct profiles of accumulation of sphingoid bases, including unknown species (Fig. 13.4). ILs’ unique stimuli could therefore be explored to activate cell wall integrity pathways in filamentous fungi and help establishing a connection of this signaling pathway with sphingolipid metabolism. Interestingly, cholinium alkanoates cannot permeabilize the plasma membrane nor cause cell wall damage, but still activate sphingolipid biosynthesis and lead to the accumulation of sphingoid bases, including unknown species. Whether these molecules are involved in the response of A. nidulans to the toxic effects of cholinium alkanoates remains unclear. The distinct profiles of accumulation of sphingoid bases displayed by each IL family further highlight the uniqueness of IL stimuli. It evidences that these compounds can be explored to further investigate the roles of sphingolipids and their intermediates as signaling molecules in filamentous fungi. Further investigations require the identification of the unknown intermediates seen to accumulate upon exposure to distinct ILs. Most of our studies focus on
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FIGURE 13.4 Chromatographic profile (high-performance liquid chromatography) of the accumulation of sphingoid bases in Aspergillus nidulans after exposure to three representative ILs: 1-decyl-methylimidazolium chloride ([C10mim]Cl), cholinium decanoate, or hexyl-(2-hydroxyethyl)-dimethylammonium bromide ([N1 1 6 2OH]Br). The control without ILs is indicated with a dashed line. Phytosphingosine (PHS) and dihydrosphingosine (DHS) are the sphingoid bases known to be produced by this fungus. x, y, and z represent unknown sphingoid bases with retention times at 8.2, 9.75, and 11.75 min, respectively (unpublished data).
A. nidulans, a model fungus that is phylogenetically closer to several emerging human-threatening fungal pathogens than S. cerevisiae. The recent demonstration that ILs might occur in nature also opens unexpected paths of their impact in biological sciences (Chen et al., 2014). This recent study revealed that a protic IL is formed during the confrontation of two ant species, Solenopsis invicta and Nylanderia fulva, probably as a form of defense mechanism against the venom of the former ant species. In line with the previous, another good example of their unexpected impacts in biological systems is the demonstration by our group that ILs alter the metabolic footprint in filamentous fungi (Petkovic et al., 2009). This study suggested that fungal cultures respond to specific ILs by changing their cell biochemistry, resulting in an altered pattern of extracellular metabolites. The hypothesis that these newly formed compounds might play uncharacterized roles in fungi deserves further investigation (Petkovic and Silva Pereira, 2012). Pathogenic and opportunistic filamentous fungi are responsible for numerous fungal infections and are emerging as causative agents in a broad diversity of clinical conditions (Brown et al., 2012). Furthermore, resistance to antifungal drugs is now an increasing risk to the lives of millions of individuals
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(Anderson, 2005). Hence, the current challenge is to better understand the biology of filamentous fungi and identify novel targets for antifungal drugs. As our group gets closer to understanding the primary effects of each IL family and move toward the identification of their specific gene targets, the vision of developing antifungal ILs and materials by taking advantage of elegant progresses in this field might become a reality. The innovative frontier research in the cross-disciplinary field of ILs might provide us unforeseen means to address these global concerns. All these new data can shed a new light on the studies of ILs toxicity. These compounds show a tremendous potential to induce unique stress responses in living organisms. Hence, ILs can in turn be explored as powerful tools in biological sciences, bringing a new boost of interest on IL research.
ACKNOWLEDGMENTS D.O.H. is grateful to Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT)ePortugal for the fellowship SFRH/BD/66396/2009. The work was partially supported by FCT Grants: PTDC/ AAC-CLI/119100/2010 and PTDC/QUIeQUI/120982/2010.
REFERENCES Abbott, A.P., Capper, G., Davies, D.L., Munro, H., Rasheed, R.K., Tambyrajah, V., 2003. Electrochemical studies of ambient temperature ionic liquids based on choline chloride. ACS Symp. Ser. 856, 439e452. Abbott, A.P., Capper, G., Davies, D.L., Rasheed, R.H., Tambyrajah, V., 2002. Quaternary ammonium zinc- or tin-containing ionic liquids: water insensitive, recyclable catalysts for DielseAlder reactions. Green Chem. 4 (1), 24e26. Abbott, A.P., Capper, G., Davies, D.L., Rasheed, R.K., Tambyrajah, V., 2001. Novel ambient temperature ionic liquids for zinc and zinc alloy electrodeposition. Trans. Inst. Met. Finish 79, 204e206. Abbott, A.P., Davies, D.L., 2000. Ionic Liquids Prepared as Low Melting Salts and Compounds of Quaternary Ammonium Halides with Metal Halides. World. Abbott, A.P., Davies, D.L., Jenkins, P., 2004. Reactions in ionic liquids based on choline chloride. Spec. Chem. 24, 36e37. Alberts, B., Johnson, J., Lewis, J., Raff, M., Roberts, K., Walter, P., 2002. Molecular Biology of the Cell, fourth ed. Garland Science, New York. Anderson, J.B., 2005. Evolution of antifungal-drug resistance: mechanisms and pathogen fitness. Nat. Rev. Microbiol. 3 (7), 547e556. Armand, M., Endres, F., MacFarlane, D.R., Ohno, H., Scrosati, B., 2009. Ionic-liquid materials for the electrochemical challenges of the future. Nat. Mat. 8 (8), 621e629. Bernot, R.J., Kennedy, E.E., Lamberti, G.A., 2005. Effects of ionic liquids on the survival, movement, and feeding behavior of the freshwater snail, Physa acuta. Environ. Toxicol. Chem. 24 (7), 1759e1765. Bradaric, C.J., Downard, A., Kennedy, C., Robertson, A.J., Zhou, Y.H., 2003. Industrial preparation of phosphonium ionic liquids. Green Chem. 5 (2), 143e152.
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Brown, G.D., Denning, D.W., Gow, N.A., Levitz, S.M., Netea, M.G., White, T.C., 2012. Hidden killers: human fungal infections. Sci. Trans. Med. 4 (165), 165rv13. Bruckner, H., Theis, C., 2010. Sequences of the polypeptide antibiotics (peptaibiotics) acretocins. J. Pept. Sci. 16, 141. Busetti, A., Crawford, D.E., Earle, M.J., Gilea, M.A., Gilmore, B.F., Gorman, S.P., Laverty, G., Lowry, A.F., McLaughlin, M., Seddon, K.R., 2010. Antimicrobial and antibiofilm activities of 1-alkylquinolinium bromide ionic liquids. Green Chem. 12 (3), 420e425. Chen, L., Mullen, G.E., Le Roch, M., Cassity, C.G., Gouault, N., Fadamiro, H.Y., Barletta, R.E., O’Brien, R.A., Sykora, R.E., Stenson, A.C., et al., 2014. On the formation of a protic ionic liquid in nature. Angew. Chem. Int. Ed. 53 (44), 11762e11765. Cho, C.W., Jeon, Y.C., Pham, T.P.T., Vijayaraghavan, K., Yun, Y.S., 2008. The ecotoxicity of ionic liquids and traditional organic solvents on microalga Selenastrum capricornutum. Ecotoxicol. Environ. Saf. 71 (1), 166e171. Cieniecka-Rosłonkiewicz, A., Pernak, J., Kubis-Feder, J., Ramani, A., Robertson, A.J., Seddon, K.R., 2005. Synthesis, anti-microbial activities and anti-electrostatic properties of phosphonium-based ionic liquids. Green Chem. 7 (12), 855e862. Cole, A.C., Jensen, J.L., Ntai, I., Tran, K.L.T., Weaver, K.J., Forbes, D.C., Davis, J.H., 2002. Novel Brønsted acidic ionic liquids and their use as dual solvent-catalysts. J. Am. Chem. Soc. 124 (21), 5962e5963. Costello, D.M., Brown, L.M., Lamberti, G.A., 2009. Acute toxic effects of ionic liquids on zebra mussel (Dreissena polymorpha) survival and feeding. Green Chem. 11 (4), 548e553. Couling, D.J., Bernot, R.J., Docherty, K.M., Dixon, J.K., Maginn, E.J., 2006. Assessing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structureeproperty relationship modeling. Green Chem. 8 (1), 82e90. Deetlefs, M., Seddon, K.R., 2006. Ionic liquids: fact and fiction. Chim. Oggi Chem. Today 24 (2), 16e23. Deetlefs, M., Seddon, K.R., 2010. Assessing the greeness of some typical laboratory ionic liquids preparations. Green Chem. 12, 17e30. Demberelnyamba, D., Kim, K.S., Choi, S.J., Park, S.Y., Lee, H., Kim, C.J., Yoo, I.D., 2004. Synthesis and antimicrobial properties of imidazolium and pyrrolidinonium salts. Bioorg. Med. Chem. 12 (5), 853e857. Dipeolu, O., Green, E., Stephens, G., 2009. Effects of water-miscible ionic liquids on cell growth and nitro reduction using Clostridium sporogenes. Green Chem. 11 (3), 397e401. Dobler, D., Schmidts, T., Klingenhofer, I., Runkel, F., 2013. Ionic liquids as ingredients in topical drug delivery systems. Int. J. Pharm. 441 (1e2), 620e627. Docherty, K.M., Kulpa, C.F., 2005. Toxicity and antimicrobial activity of imidazolium and pyridinium ionic liquids. Green Chem. 7 (4), 185e189. Dong, Q., Muzny, C.D., Kazakov, A., Diky, V., Magee, J.W., Widegren, J.A., Chirico, R.D., Marsh, K.N., Frenkel, M., 2007. ILThermo: a free-access web database for thermodynamic properties of ionic liquids. J. Chem. Eng. Data 52 (4), 1151e1159. Evans, K.O., 2008. Supported phospholipid bilayer interaction with components found in typical room-temperature ionic liquidsda QCM-D and AFM study. Int. J. Mol. Sci. 9 (4), 498e511. Faral-Tello, P., Liang, M., Mahler, G., Wipf, P., Robello, C., 2014. Imidazolium compounds are active against all stages of Trypanosoma cruzi. Int. J. Antimicrob. Agents 43 (3), 262e268. Ferreira, R., Garcia, H., Sousa, A.F., Petkovic, M., Lamosa, P., Freire, C.S.R., Silvestre, A.J.D., Rebelo, L.P.N., Pereira, C.S., 2012. Suberin isolation from cork using ionic liquids: characterisation of ensuing products. New J. Chem. 36 (10), 2014e2024.
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Ferreira, R., Garcia, H., Sousa, A.F., Guerreiro, M., Duarte, F.J.S., Freire, C.S.R., Calhorda, M.J., Silvestre, A.J.D., Kunz, W., Rebelo, L.P.N., et al., 2014. Unveiling the dual role of the cholinium hexanoate ionic liquid as solvent and catalyst in suberin depolymerisation. RSC Adv. 4 (6), 2993e3002. Fredenhagen, A., Molleyres, L.P., Bohlendorf, B., Laue, G., 2006. Structure determination of neoefrapeptins A to N: peptides with insecticidal activity produced by the fungus Geotrichum candidum. J.
Toxicity of Ionic Liquids: Past, Present, and Future Diego O. Hartmann, Cristina Silva Pereira Universidade Nova de Lisboa, Instituto de Tecnologia Quı´mica e Biolo´gica Anto´nio Xavier, Oeiras, Portugal
Ionic liquids (ILs) have been described as molten salts that have, by definition, a melting point below 100 C (Stark and Seddon, 2007). They are entirely ionic in nature and usually consist of a large asymmetrical organic cation associated with a polyatomic anion that may be either organic or inorganic. In the design of ILs, the asymmetry of the cation or anion is preferred over symmetrical species, since the former do not tend to pack so easily, therefore rendering salts with a lower melting point (Stark and Seddon, 2007). Structural modifications can be made either to the anion, to the cation, or in substituents on these two species. Therefore, millions of formulations become possible and, by altering their cationic or anionic components, their properties can be tailored (Plechkova and Seddon, 2007; Stark and Seddon, 2007). Their physical and chemical properties (eg, melting point, viscosity, solubility, or hydrophobicity and chemical polarity or hydrogen bonding ability, respectively) can be tuned to best suit a specific process. The tunability of such properties rendered ILs an excellent choice as alternative solvents in many chemical processes. They already found applications in many reactions (Plechkova and Seddon, 2008), as solvents (Holbrey and Seddon, 1999; Plechkova and Seddon, 2008) and catalysts (Cole et al., 2002), and show great potential in electrochemistry (Armand et al., 2009; Sakaebe et al., 2007). ILs’ physical and chemical properties, together with their negligible vapor pressure and bulk nonflammability, suggested them as potential green solvents (Deetlefs and Seddon, 2006, 2010; Dong et al., 2007). Their negligible volatility distinguishes them from traditional organic solvents, reducing risk of atmospheric pollution. However, the potential release of IL vapors should be considered when used at elevated temperatures (Ranke et al., 2007a,b). Nonetheless, soil and water contamination, either by accidental spills or waste disposal, constitutes a serious hazard. The ecotoxicological assessment of this class of compounds should be considered a priority, and has been taken into Ionic Liquids in Lipid Processing and Analysis. http://dx.doi.org/10.1016/B978-1-63067-047-4.00013-1 Copyright © 2016 AOCS Press. Published by Elsevier Inc. All rights reserved.
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account for the conscious design of novel ILs (Petkovic et al., 2011; Petkovic and Silva Pereira, 2012).
13.1 PAST: EXTENSIVE STUDIES ON IONIC LIQUID TOXICITY Interest in IL toxicity began in the early 2000s. Pernak et al. (2001a,b) developed new cationic surfactants with benzimidazolium or pyridinium cores and evaluated their antimicrobial properties (minimal inhibitory and lethal concentrations against bacterial and yeast strains relevant for human health). The evaluation of the antimicrobial activity of 1-alkoxymethyl-(3-nicotionylaminomethyl)benzimidazolium, 1-alkoxymethyl-3-(1-benzimidazolmethylamino)pyridinium, 1-alkoxymethyl-3-[1-(benzotriazol-1-yl)methylamino]pyridinium chlorides, and N,N0 -bis[3-(1-alkoxymethyl)pyridinium chloride]methylenediamine revealed that their toxicity increased with the elongation of the alkoxy chain, which varied between 2 and 12 carbon atoms. This constituted one of the first systematic studies on the toxicity of ILs, and served afterward as a basis for evaluating the toxicity of many newly synthesized imidazolium- and pyridinium-based compounds (Fig. 13.1), including 1-alkoxymethylcarbamoylpyridinium chlorides (Pernak et al., 2001a,b), 1-alkyl-3-methylimidazolium chlorides, and bromides; 1-alkyl-3-hydroxyethyl2-methylimidazolium chlorides (Demberelnyamba et al., 2004), 1-alkoxymethyl-3-methylimidazolium chlorides, tetrafluoroborates and hexafluorophosphates (Pernak et al., 2003), 1,3-dialkoxymethylimidazolium chlorides (Pernak et al., 2004a,b,c), 1-alkyl- and 1-alkoxy-methylimidazolium lactates (Pernak et al., 2004a,b,c), 1-alkoxymethyl-3-hydroxypyridinium, and 1-alkoxymethyl-3-dimethylaminopyridinium chlorides (Pernak and Branicka, 2003). The data revealed a clear trend toward a stronger toxic effect of ILs with the increase in the length of the alkyl or the alkoxy side chain. Further studies revealed that the incorporation of an oxygen atom in the side chain significantly reduced the toxicity of the ILs (Morrissey et al., 2009). Symmetrical chains in positions R1 and R3 of the imidazolium ring, however, resulted in lower toxicity, probably due to steric effects with the cell surface (Pernak et al., 2004a,b,c). The effect of anions on the observed toxicities seemed to be secondary to the effect of the cations, but their broad diversity did not allow a conclusive analysis. These initial observations inspired an escalating number of studies on toxicity of several ILs, expanding the research for other biological models, particularly model eukaryotic organisms. For example, the toxicity of ILs with 1-alkyl-3-methylimidazolium as cation were analyzed against leukemia and glioma rat cell lines (Ranke et al., 2004), human HeLa cell lines (Stepnowski et al., 2004), and the worm Caenorhabditis elegans (Swatloski et al., 2004). Additionally, the toxicity of pyridinium-based ILs has been analyzed using freshwater snail Physa acuta (Bernot et al., 2005), the zebra mussel Dreissena polymorpha (Costello et al., 2009), and green algae Pseudokirchneriella subcapitata (Pham et al., 2008), focusing on the effect of the alkyl side chain
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(B)
(A)
R2 +
+
R1
N
N
R3
N
R1
(D)
R
(E)
(F)
+
+
N
R1
(G)
+
N
R2
R1
R2
(H)
+
R2 N
+
O
N
R2
R1
+
N
R2
R1
(C)
R2 R3 R4
P
R1
R3 R4
FIGURE 13.1 Common IL cation cores and some of their possible structural modifications: (A) imidazolium, (B) pyridinium, (C) quinilinium, (D) pyrrolidinium, (E) piperidinium, (F) morpholinium, (G) quaternary ammonium, and (H) quaternary phosphonium cations.
of the pyridinium cation. The effects of imidazolium- and pyridinium-based ILs in the bioluminescence of the bacterium Vibrio fischeri were also analyzed (Docherty and Kulpa, 2005). Bacterial bioluminescence is directly linked to cellular respiration, so decrease in luminescence is indicative of toxicity. Other cationic head groups were also evaluated on their toxic effects, such as the aromatic quinolinium-based cations (Fig. 13.1). Despite rarely being studied, these ILs showed great antimicrobial properties and cytotoxicity potential that increased with elongation of substituted alkyl chain (Busetti et al., 2010; Ranke et al., 2007a,b). The effects of ILs with nitrogen-containing alicyclic cations, namely pyrrolidinium, piperidinium, and morpholinium (Fig. 13.1), were also investigated; for example, their aquatic toxicity against the zebrafish Danio rerio (Pretti et al., 2006) and their cytotoxicity against mammal cell lines (Kumar et al., 2009; Stolte et al., 2007a,b). It was observed that increasing numbers of carbon atoms in an alicyclic ring generally increases toxicity; for example, the piperidinium cation (six-member rings) was more toxic than the pyrrolidinium cation (five-member rings) (Kumar et al., 2009). The morpholinium ILs, due to incorporation of an
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oxygen atom in the ring, were the least toxic of these alicyclic cations. Furthermore, the nonaromatic head groups appeared to be generally less toxic than their aromatic equivalents (Kumar et al., 2009; Stolte et al., 2007a,b). Their lower toxicity, relative to the corresponding aromatic rings, was generally evident in the bioassays with models such as V. fischeri, the algae Scenedesmus vacuolatus, and the aquatic plant Lemna minor (Stolte et al., 2007a,b). As observed for imidazolium- and pyridinium-based ILs, the length of the alkyl chains in the cation played a major role in toxicity of piperidinium- and pyrrolidinium-based ILs (Kumar et al., 2009; Ranke et al., 2007a,b). Anions have been considered to have an unpredictable effect, largely reflecting the lack of systematization in their ecotoxicological assessment. Quaternary ammonium salts (Fig. 13.1) have been largely studied and explored in numerous applications, such as disinfectants, surfactants, antistatic agents, and catalysts (Jones, 2001). Their properties depend on chain length and functional groups of anion (Ying, 2006). Pernak and coauthors were the first to consider their antimicrobial activity against relevant clinical strains, which was, as expected, governed by length of the alkoxy and alkyl side chains (Pernak and Chwała, 2003; Pernak and Feder-Kubis, 2005). The toxicity of ammonium ILs against other biological models was also tested, including bioluminescent bacterium V. fischeri (Couling et al., 2006), fresh water crustacean Daphnia magna, and algae P. subcapitata (Pretti et al., 2009; Wells and Coombe, 2006), zebrafish (Pretti et al., 2009), and cell lines (Ranke et al., 2007a,b). The same toxicity trends previously described for other cationic head groups were observed; the highest toxicity was detected for those with longest alkyl chains and could be well correlated with the cation lipophilicity. Some of the most interesting groups of quaternary ammonium-based ILs are those containing the cholinium cation (2-hydroxyethyl-trimethylammonium). Its combination with benign anions constituted a major advance in the design of biocompatible ILs (Abbott et al., 2001, 2002, 2003, 2004; Abbott and Davies, 2000; Fukaya et al., 2007; Pernak et al., 2007; Renshaw, 1909). The low toxicity of many cholinium ILs has been demonstrated, including those carrying as anions; for example, saccharinate and acesulfamate (Nockemann et al., 2007), dimethylphosphate (Dipeolu et al., 2009), phosphate-based anions (Weaver et al., 2010), lactates (Petkovic et al., 2009), and alkanoates (Petkovic et al., 2010). The last were analyzed by our group for the first time against filamentous fungi of the genus Penicillum, and included a range of linear alkanoate anions and two structural isomers (Petkovic et al., 2010). Their toxicity increased with the elongation of the anion, with the branched isomers less toxic than corresponding linear ones with an equal number of carbon atoms. These cholinium alkanoates also displayed high biodegradability potential and great solvent ability (Garcia et al., 2010). These findings have been strengthened by other groups, which also evaluated their cytotoxicity (Klein et al., 2013). The research focus on toxicity of ILs is moving toward other cationic groups aside imidazolium- and pyridinium-based ones, such as the quaternary
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phosphonium cations (Fig. 13.1). These ILs are currently produced in tonne quantities, particularly by Cytec Industries Inc. (Bradaric et al., 2003), further highlighting the need for their ecotoxicological assessment, which is still seldom investigated. Some halides (eg, with the tetrabutylphosphonium cation) showed levels of toxicity against the freshwater snail P. acuta that were comparable to imidazolium-based ILs carrying the same chain length (Bernot et al., 2005). The antimicrobial properties of a series of alkyltrihexylphosphonium halides demonstrated a significant role of the structure of the cation in their toxicity, since the antimicrobial activity decreased for the longest alkyl chains (8e14 carbon atoms) (Cieniecka-Rosłonkiewicz et al., 2005). The apparently high toxicity of tetraalkylphosphonium halides against V. fischeri, and D. magna (Couling et al., 2006; Wells and Coombe, 2006), and P. subcapitata (Cho et al., 2008; Wells and Coombe, 2006) was also demonstrated, as well as their cytotoxicity against human and rat cell lines (Ranke et al., 2007a,b, Wang et al., 2007). The lack of systematization in these studies, however, did not allow a conclusive rationalization, a general problem when evaluating the toxicity of the highly diverse groups of ILs (Petkovic et al., 2011; Petkovic and Silva Pereira, 2012). These initial studies were essential to establish the foundations for evaluating the ecotoxicity of ILs, particularly for understanding how the cationic or anionic head groups and their structural modifications impact toxicity. Despite the massive amount of significant data produced so far, evidence of their specific mechanisms of toxicity is still largely lacking. In-depth knowledge of their toxicity is critical for developing safer IL based-processes and may provide unforeseen opportunities in IL frontier research.
13.2 PRESENT: UNDERSTANDING THE MOLECULAR BASIS OF IONIC LIQUID TOXICITY The hydrophobic interactions between ILs and biological membranes have been proposed as a main mechanism of toxicity of ILs, largely supported by the observation that their toxic effects increase with the elongation of the side chains. This nonspecific toxicity, called baseline toxicity or narcosis (Mackay et al., 2009), appears to be correlated with 1-octanol/water partition coefficients. This is suggestive of an increase in either the cation or anion lipophilicity with elongation of the side chains (Stolte et al., 2007a,b), leading to greater probability of interaction with biological membranes. Liposomes (ie, phospholipid vesicles) were used to assess the effects of some ILs in cellular membranes (Schaffran et al., 2009). As an example, the 1-octyl-3-methylimidazolium cation was demonstrated to cause severe disruptions in a supported phospholipid bilayer (Evans, 2008). Molecular dynamics simulation studies also tried to demonstrate the interactions of ILs with artificial lipid bilayers (Klein et al., 2013; Ruegg et al., 2014). These elegant models are, however, unable to reproduce the complexity of
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the heterogeneous lipid bilayers of biological membranes. Therefore, real biological systems, with all their intrinsic complexity, are required to evaluate the true effects of ILs. The lack of systematic studies on membrane permeabilization as a mechanism for toxicity of ILs was the driving force of our work. We pioneered the use of filamentous fungi ecotoxicological assessment of ILs (Petkovic et al., 2009). They were shown to tolerate, in general, very high concentrations of ILs. Moreover, their high catabolic capacities render them as great candidates for biodegradation studies. We have successfully established all the methodologies necessary to evaluate the effects of ILs against filamentous fungi, excellent model organisms for moving beyond the state-of-the-art on ILs toxicity. The first ILs subjected to in-depth investigation of their mechanisms of toxicity was a series of highly toxic alkyltributylphosphonium chlorides, [P4 4 4 n]Cl, where n ¼ 1, 4e8, 10, 12, or 14. A fluorescence microscopy assessment demonstrated that these ILs were able to permeabilize the plasma membrane of the spores of the model filamentous fungus Aspergillus nidulans (Fig. 13.2) (Petkovic et al., 2012). Their toxicity was observed to increase with systematic elongation of one alkyl substituent, consistent with the increase in their 1-octanol/water partition coefficient. The data clearly suggested that the toxicity of [P4 4 4 n]Cl, where n 4, is ruled by direct interaction with the plasma membrane. Further confirmation of these effects was attained by analyzing IL effects at a molecular level, in particular by measuring the expression levels of genes involved in plasma membrane biosynthesis in A. nidulans (Hartmann and Silva Pereira, 2013). The data suggested that the fungus alters the plasma membrane fluidity in response to the membrane permeabilization provoked by the ILs. Both fluorescence microscopy and gene expression analysis were proven to be powerful and rapid tools to investigate in vivo the ability of ILs to permeabilize biological membranes. Accordingly, these methods were used to investigate the toxic effects of other important families of ILs. The imidazolium-based ILs are probably the most investigated group and one of the first to find application on an industrial scale (Plechkova and Seddon, 2008). The cholinium-based ILs are currently gaining great interest for the development of biocompatible ILs, mainly due to the benign nature of the cholinium cation (Klein et al., 2013; Pernak and Chwała, 2003; Pernak et al., 2007; Petkovic et al., 2010). We have chosen representative families of each of these IL groups, namely 1-alkyl-3-methylimidazolium chlorides ([Cnmim]Cl, where n ¼ 2, 4, 6, 8, or 10) and cholinium alkanoates (anion ¼ ethanoate, butanoate, hexanoate, octanoate, or decanoate), to perform a systematic investigation of their toxic effects and mechanisms of toxicity. As previously observed for the phosphonium-based ILs, both 1-alkyl-3-methylimidazolium chlorides and cholinium alkanoates present the same trend of toxicity against A. nidulans: their toxicity increased with the elongation of the alkyl
FIGURE 13.2 Fluorescence microscopy of asexual spores of Aspergillus nidulans exposed to alkyltributylphosphonium chlorides ([P4 4 4 n]Cl, n ¼ 1, 4, 8, or 12) for 1 h and stained with propidium iodide, which only stains cells with permeabilized membranes. The left column shows the total number of spores and the right column shows membrane-damaged spores. The percentage of damaged cells increases with the elongation of the alkyl chain in the cation. Scale bar: 20 mm. Adapted from Petkovic, M., Hartmann, D.O., Adamova´, G., Seddon, K.R., Rebelo, L.P.N., Silva Pereira, C., 2012. Unravelling the mechanism of toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans conidia. New J. Chem. 36 (1), 56e63.
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chain either in the cation or the anion, respectively. However, while imidazolium-based ILs appear to be toxic and recalcitrant to degradation, cholinium alkanoates present low toxicity and are readily biodegradable (Petkovic et al., 2010). Furthermore, 1-alkyl-3-methylimidazolium chlorides with long alkyl chains ([Cnmim]Cl, where n 6) were able to permeabilize the fungal plasma membrane, leading to cell death, while cholinium alkanoates could not cause such an effect (Hartmann et al., 2015). These differences were most likely due to the nature of each IL family. The plasma membrane is charged along its surface, due to the different composition of neutral and negatively charged head groups of its composing phospholipids (Alberts et al., 2002). Toxicity of 1-alkyl-3-methylimidazolium chlorides seems to closely relate to the elongation of the alkyl chain in the cation, while for cholinium alkanoates, increasing number of carbons in the anion appears to rule their toxicity. Most probably, the negatively charged phospholipids assist the interaction of the imidazolium-based cations with the plasma membrane, allowing its permeabilization, while the anions from cholinium alkanoates are likely repelled. The cholinium cation, on the other hand, can interact with the surface of the plasma membrane, but its bulky nature does not allow membrane permeabilization. Elongating one of the alkyl chains of the cholinium cation, however, enables plasma membrane permeabilization. This could be observed for a series of alkyl-(2-hydroxyethyl)-dimethylammonium bromides ([N1 1 n 2OH]Br, where n ¼ 2, 4, 6, 8, 10 or 12) (Hartmann et al., 2015). The analyses of the toxicity of these cholinium derivatives reveal that this family of ILs presented membrane-permeabilizing effects but maintains the low toxicity and the biodegradability of the archetype cholinium-based ILs. These observations constitute a unique asset for the development of new biocompatible ILs: linear elongation of alkyl chains should be preferentially introduced in the anions, in order to avoid permeabilization of biological membranes. Interaction with the plasma membrane and its disruption is most likely a nonspecific mechanism of toxicity of ILs, probably observed for all cellular organisms. In addition to the plasma membrane the fungal cell envelope comprises another complex structure, the cell wall. The cell wall of A. nidulans presents an inner layer composed of polysaccharides, mainly chitin, 1,3-b-glucans, and 1,3-a-glucan (de Groot et al., 2009). The glucan network serves as a support for an outer layer of mannoproteins. The presence of these mannoproteins confers an overall negative charge to the cell wall that might also have an implication on the effects of ILs in fungi. Employing fluorescence microscopy, gene expression analysis, and scanning electron microscopy, we analyzed the ability of ILs to cause damage to the fungal cell wall and observed that alkyltributylphosphonium chlorides, in fact, damage this cellular structure, regardless of the alkyl chain length (Fig. 13.3) (Hartmann and Silva Pereira, 2013; Petkovic et al., 2012). Other ILs with long
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FIGURE 13.3 Scanning electron microscopy analysis of asexual spores of Aspergillus nidulans exposed to alkyltributylphosphonium chlorides ([P4 4 4 n]Cl, n ¼ 1, 8, or 12) for 2 h. Images reveal that ILs cause severe damage to the cell wall and surface of the cells. Micrographs on the left and on the right have 5000 and 15,000 magnification, respectively. Scale bars: 1 mm. Adapted from Petkovic, M., Hartmann, D.O., Adamova´, G., Seddon, K.R., Rebelo, L.P.N., Silva Pereira, C., 2012. Unravelling the mechanism of toxicity of alkyltributylphosphonium chlorides in Aspergillus nidulans conidia. New J. Chem. 36 (1), 56e63.
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alkyl chains in the cation, namely 1-alkyl-3-methylimidazolium chlorides and alkyl-(2-hydroxyethyl)-dimethylammonium bromides, also induce cell wall damage (unpublished data). Cholinium alkanoates, however, do not cause such effects. It seems possible that the negative charge of the cell wall also played a pivotal role in the effects of ILs upon this cellular structure. Additional studies were undertaken by our group to further investigate the mechanisms used by filamentous fungi to tolerate ILs. We analyzed the mycelial proteome of two model fungi upon exposure to 1-ethyl-3methylimidazolium chloride and cholinium chloride, which were selected since they present opposite toxicity and recalcitrance toward fungi (Martins et al., 2013). Data obtained showed that numerous stress-responsive proteins (eg, anti-ROS defense proteins and drug transporter proteins) as well as several critical biological processes and/or pathways were affected by either IL. Among other changes, these compounds altered developmental program in both fungi, for example, promoting the formation of Hu¨lle cells (involved in sexual development of the fungus) or asexual sporulation, and led to accumulation of osmolytes, namely glycerol in Neurospora crassa and betaine in A. nidulans. This is consistent with the observation that addition of betaine to the bacterium Enterobacter lignolyticus growth media supplemented with 0.375 M of 1-ethyl-3-methylimidazolium chloride increased biomass production (Khudyakov et al., 2012). Interestingly, the proteomic study also shed light on the mechanism of toxicity of cholinium-based ILs. Major uptake of the cholinium cation led to the production of the toxic compound cyanide, which might be a cause of the observed growth inhibition provoked by this group of ILs (Martins et al., 2013). Another remarkable finding was the accumulation of a protein likely involved in the biosynthesis of unusual amino acids in N. crassa (unpublished data). In some fungi, these rare amino acids are involved in the formation of unusual metabolites, such as neoefrapeptins and acretocins, which usually show broad antimicrobial activity (Bruckner and Theis, 2010; Fredenhagen et al., 2006). Further tests are ongoing to confirm the presence of metabolites potentially carrying interesting biological activity. These results clearly demonstrate the importance of unraveling the mechanisms of toxicity of ILs at a molecular level, revealing that their full potential remains largely overlooked.
13.3 FUTURE: MOVING BEYOND TOXICITY TOWARD NEW POTENTIAL APPLICATIONS From the knowledge obtained by the ecotoxicological assessment of ILs and their mechanisms of toxicity, two paths are possible. The first is the development of new green, safer ILs and their application in environmentally friendly processes. As an example, the biocompatible and biodegradable cholinium alkanoates developed by our group (Petkovic et al., 2010) have been observed to have a great solvent ability toward the plant polymer suberin
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(Ferreira et al., 2012; Garcia et al., 2010; Petkovic et al., 2010). Cholinium hexanoate was successfully employed in the extraction of suberin from cork where it plays the dual roles of solvent and mild catalyst (Ferreira et al., 2014). This ensured the preservation of the native structure of the polymer, which allowed, for the first time, the formation of suberin films with antibacterial properties (Garcia et al., 2014). Moreover, deep knowledge on IL toxicity inspired the development of engineered bacteria carrying phenotypes with increased resistance to particular ILs in order to promote their efficient use in biorefineries and biofuel production (Khudyakov et al., 2012; Ruegg et al., 2014). The second possible path is to explore ILs in biological applications, in which their unique toxic properties become useful. A good example of their early applications was the innovative use of 1-alkoxymethyl-3methylimidazolium tetrafluoroborate in embalming and tissue preservation (Majewski et al., 2003), exploring their antimicrobial activity as a replacement for the highly volatile and toxic formalin, and as a wood preservative (Pernak et al., 2004a,b,c). The potential use of ILs as drugs for important etiological agents is also under current investigation. For example, imidazolium-based ILs have been explored for their potential in combating Plasmodium falciparum (Vlahakis et al., 2010) and Trypanosoma cruzi (Faral-Tello et al., 2014), the causative agents of malaria and Chagas disease, respectively. However, their cytotoxicity still hampers their clinical usage. A better example is choline-geranate that has been proposed as a coadjutant in the treatment of wound infections, due to its antimicrobial activity, minimal toxicity to epithelial cells as well as skin, and effective permeation enhancement for drug delivery (Zakrewsky et al., 2014). Probably one of the most interesting and promising applications of ILs is their combination with active pharmaceutical ingredients (APIs) (Marrucho et al., 2014). These pure liquid salt forms of active pharmaceutical ingredients (API-ILs) are considered a novel strategy to overcome existing problems with solid-state APIs. This strategy potentially allows the liquefaction of the drug and when combined with adequate ionic counterparts can enhance its delivery. Several drugs have already been successfully synthesized in their API-IL form, such as the antiinflammatory ibuprofen, the antibiotic amoxicillin, and the anesthetic procaine (Marrucho et al., 2014). Progress in this field requires full compliance of the new API-ILs with the extant legislation. ILs have also been explored to increase the solubility of insoluble drugs and enhance their topical and transdermal delivery (Dobler et al., 2013; Shamshina et al., 2013). Our group has ongoing side projects that aim at using IL formulations to tune the solubility of well-established cytotoxic antifungal drugs, while establishing proof-of-concept that their mechanism of activity remains unaltered. Rather unexplored is the potential of ILs to induce metabolic alterations and stress responses in living organisms that might be of great interest in biological sciences. Our studies on the mechanisms of toxicity of ILs revealed
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that some ILs not only cause plasma membrane permeabilization and cell wall damage, but also seem to induce alteration in lipid metabolism in filamentous fungi, more specifically in the synthesis of sphingolipids (unpublished data). Fungi respond to damage to the cell wall by activating several genes involved in its biosynthesis, creating conditions that allow them to reestablish its integrity. This response is regulated by a signaling cascade better understood in the yeast Saccharomyces cerevisiae, called cell wall integrity pathway (Levin, 2005). This pathway is also known to be present in a variety of filamentous fungi, but many knowledge gaps in their constitution and regulation call for further investigation. It is thought that A. nidulans possesses an alternative pathway for cell wall integrity regulation, still uncharacterized (Fujioka et al., 2007). Exposure to alkyltributylphosphonium chlorides, as an example, led to cell wall damage and was believed to induce the activation of an alternative cell wall integrity pathway (Hartmann and Silva Pereira, 2013). This scientific hypothesis is tremendously exciting, especially when considering the significance of this pathway as a potential pathogen-specific target for the development of new antifungal drugs. Importantly, there are only three major classes of antifungal drugs in clinical use and the current pace of drug development is inadequate to fight the threat of emerging fungal pathogens and multidrug resistance (Ostrosky-Zeichner et al., 2010). Interfaces of the cell wall integrity pathway with other signaling pathways in S. cerevisiae have been observed, including the sphingolipid metabolism (Levin, 2005). This, however, has not yet been elucidated in the filamentous fungus A. nidulans. We have observed that some ILs that cause membrane permeabilization and cell wall damage also activate the sphingolipid biosynthetic pathway and induce accumulation of certain sphingolipid intermediates, such as sphingoid bases (unpublished data). It is possible that these molecules are acting as second messengers for the response to cell wall stress induced by ILs. Curiously, each family of ILs tested seemed to induce distinct profiles of accumulation of sphingoid bases, including unknown species (Fig. 13.4). ILs’ unique stimuli could therefore be explored to activate cell wall integrity pathways in filamentous fungi and help establishing a connection of this signaling pathway with sphingolipid metabolism. Interestingly, cholinium alkanoates cannot permeabilize the plasma membrane nor cause cell wall damage, but still activate sphingolipid biosynthesis and lead to the accumulation of sphingoid bases, including unknown species. Whether these molecules are involved in the response of A. nidulans to the toxic effects of cholinium alkanoates remains unclear. The distinct profiles of accumulation of sphingoid bases displayed by each IL family further highlight the uniqueness of IL stimuli. It evidences that these compounds can be explored to further investigate the roles of sphingolipids and their intermediates as signaling molecules in filamentous fungi. Further investigations require the identification of the unknown intermediates seen to accumulate upon exposure to distinct ILs. Most of our studies focus on
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FIGURE 13.4 Chromatographic profile (high-performance liquid chromatography) of the accumulation of sphingoid bases in Aspergillus nidulans after exposure to three representative ILs: 1-decyl-methylimidazolium chloride ([C10mim]Cl), cholinium decanoate, or hexyl-(2-hydroxyethyl)-dimethylammonium bromide ([N1 1 6 2OH]Br). The control without ILs is indicated with a dashed line. Phytosphingosine (PHS) and dihydrosphingosine (DHS) are the sphingoid bases known to be produced by this fungus. x, y, and z represent unknown sphingoid bases with retention times at 8.2, 9.75, and 11.75 min, respectively (unpublished data).
A. nidulans, a model fungus that is phylogenetically closer to several emerging human-threatening fungal pathogens than S. cerevisiae. The recent demonstration that ILs might occur in nature also opens unexpected paths of their impact in biological sciences (Chen et al., 2014). This recent study revealed that a protic IL is formed during the confrontation of two ant species, Solenopsis invicta and Nylanderia fulva, probably as a form of defense mechanism against the venom of the former ant species. In line with the previous, another good example of their unexpected impacts in biological systems is the demonstration by our group that ILs alter the metabolic footprint in filamentous fungi (Petkovic et al., 2009). This study suggested that fungal cultures respond to specific ILs by changing their cell biochemistry, resulting in an altered pattern of extracellular metabolites. The hypothesis that these newly formed compounds might play uncharacterized roles in fungi deserves further investigation (Petkovic and Silva Pereira, 2012). Pathogenic and opportunistic filamentous fungi are responsible for numerous fungal infections and are emerging as causative agents in a broad diversity of clinical conditions (Brown et al., 2012). Furthermore, resistance to antifungal drugs is now an increasing risk to the lives of millions of individuals
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(Anderson, 2005). Hence, the current challenge is to better understand the biology of filamentous fungi and identify novel targets for antifungal drugs. As our group gets closer to understanding the primary effects of each IL family and move toward the identification of their specific gene targets, the vision of developing antifungal ILs and materials by taking advantage of elegant progresses in this field might become a reality. The innovative frontier research in the cross-disciplinary field of ILs might provide us unforeseen means to address these global concerns. All these new data can shed a new light on the studies of ILs toxicity. These compounds show a tremendous potential to induce unique stress responses in living organisms. Hence, ILs can in turn be explored as powerful tools in biological sciences, bringing a new boost of interest on IL research.
ACKNOWLEDGMENTS D.O.H. is grateful to Fundac¸a˜o para a Cieˆncia e a Tecnologia (FCT)ePortugal for the fellowship SFRH/BD/66396/2009. The work was partially supported by FCT Grants: PTDC/ AAC-CLI/119100/2010 and PTDC/QUIeQUI/120982/2010.
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