Team:Bielefeld-CeBiTec/Toxicity Theory

Toxicity

Short Summary

On the one hand, gold, silver, copper and iron ions are of interest due to their chemical properties. On the other hand, some of the ions possess toxic properties for the cell (Ochoa-Herrera et al., 2011; Shareena et al., 2015; Zhao & Stevens, 2009). Therefore, the toxicity of the different ions of the metals needs to be included into consideration when planning the uptake and processing of these ions as a substrate for metal nanoparticle (NP) production by bacteria. Not only is the toxicity of the dissolved ions of interest but also the potential toxic effect of nanoparticles on the cell.

Gold

Gold nanoparticles (AuNPs) are popular in many scientific applications, e.g. the transfection of human cells (Chang et al., 2008). However, there are several contradicting reports regarding their toxicity (Brust et al., 1995; Caruntu et al., 2002; Zhang et al., 2015). AuNPs in the form of rod-shaped nanoparticles ("nanorods") were identified as non-toxic, solely the coating agent was found to be toxic (Alkilany et al., 2009). AuNPs in general do not possess toxic properties. Reported toxic effects of AuNPs resulted from poor purification of the AuNPs from Au(III) (Shareena Dasari, 2015).
However, gold ions, such as Au(I) and Au(III), have toxic properties. Shareena Dasari et al. demonstrated in 2015 that exposure to Au(I) and Au(III) inhibits the growth of nonpathogenic Escherichia coli as seen in figure 1. Furthermore, they determined the half maximal inhibitory concentrations (IC₅₀) for both ions in the examined buffers as seen in table 1, clearly indicating that gold ions possess toxic properties to E. coli. The toxic effect of gold ions is due to its ability to oxidative cleavage of peptide and protein disulfide bonds (Witkiewicz & Shaw, 1981). Since gold ions are toxic to the NP producing cell, the reduction to AuNPs is desirable, however the stress to the cell and its inhibitory effect have to be investigated.

**Figure 1:** Concentration dependent growth inhibition by Au(I) and Au(III) ions on multi-drug resistant *E. coli* (BAA-1161) according to Shareena Dasari (2015).

Silver

Silver nanoparticles (AgNPs) are known to possess toxic properties (Colman et al., 2013). The toxicity of AgNPs varies in response to the shape and size of the AgNPs. The toxicity increases significantly when the AgNPs are smaller than 10 nm (Ivask et al., 2014). It has been demonstrated that AgNPs exhibit toxic effects in bacteria at environmentally relevant concentrations (Colman et al., 2013). The growth inhibition is dependent on the concentration of the AgNPs. The minimal inhibitory concentration (MIC) for AgNPs in E. coli is considered to be between 3.3 and 6.6 nM (Kim et al., 2007). The exposure to AgNPs results in an apoptosis-like response of the cell. First, the membrane is depolarized by AgNPs, generating reactive oxygen species (ROS), such as hydroxyl peroxide, hydroxyl radicals and superoxide anions. These ROS cause oxidative stress and lead to the fragmentation of DNA. Simultaneously, calcium accumulates in the cytoplasm of the cell. An increased concentration of calcium results in the inversion of phosphatidylserine in the membrane. All of these processes ultimately lead to the activation of bacterial caspase-like proteins (Bortner & Cidlowski, 2007; Yun & Lee, 2017). However, there are considerations whether the AgNPs are toxic or whether the toxic effect is due to ions dissolving from the nanoparticles (Hwang et al., 2008).
The toxic effects of silver salts have been known since antiquity. Even Hippocrates recognized its antimicrobial properties in water (Magner, 1992). The toxic effect of silver ions relies on the same chemical features as those of the AgNPs. When being exposed to silver ions, the bacterium experiences several forms of oxidative stress, mainly ROS which arise from Fenton chemistry. Ag(I) disrupts metabolic pathways that drive Fenton chemistry and lead to the overproduction of hydroxyl radicals and cell death (Morones-Ramirez et al., 2013). At a concentration of 18.9 µM, all bacterial growth comes to an end (Zhao & Stevens, 1998).
Silver is toxic in both of its forms: as a nanoparticle and as dissolved ions. Therefore, an approach to decrease the toxic effect on the cell is needed.

Copper

Copper is known for its toxic and bactericidal properties and is often used as an antimicrobial agent in form of copper nanoparticles (CuNPs) (Cioffi et al., 2005). Recent studies revealed that the inactivation and bactericidal activity works in a size-dependent manner. The smaller a CuNP is the higher is its inhibitory potential to bacteria. Furthermore, the toxicity is probably elicited in a strain-specific manner in E. coli obtained from different sources (Alum et al., 2018). CuNPs’ toxic properties appear to result from oxidative stress and protein damage, DNA damage and membrane damage. The generation of hydroxyl peroxide by CuNPs is assumed to be the reason for the toxicity of CuNPs. Li et al. reported 2013 the release of copper and the production of Cu(I) ions.
Copper is considered to be essential in small amounts to both human and bacteria (Burgess et al., 1999). It plays an important role in electron-transfer reactions (Kaplan & Lutsenko, 2009). Yet copper ions can induce cell death and exhibit toxic effects. This is due to copper ions interfering with the cell proteins or enzymes by chelating sulfhydryl groups and peroxidizing the lipids of the cell membrane (Yeager, 1991). A broad variety of microorganisms, such as E. coli, are inhibited in growth or killed by exposure to Cu(II) (Ochoa-Herrera et al., 2011). Furthermore, copper ions can interact with oxygen by catalyzing Haber-Weiss and/or Fenton reactions. This results in the generation of ROS which can ultimately damage biomolecules (Halliwell, 2007; Halliwell & Gutteridge, 2015).
Copper is toxic in both of its forms: as a nanoparticle and as dissolved ions. Therefore, an approach to decrease the toxic effect on the cell is needed.

Iron

Iron is crucial for certain biological functions and necessary for the cell in order to work properly. Especially when it comes to electron transfer reactions iron as a cofactor is critical for the formation of biomolecules (Zhang, 2014). However, free non-bound iron bears risks for the cell. Iron regularly appears as a ferrous (Fe(II)) or as a ferric ion (Fe(III)) and is able to accept an electron from neighboring molecules which leads to the generation of ROS and causes damage to cellular components. The generation of ROS occurs by Fenton and Haber-Weiss reactions (Kehrer, 2000; Winterbourn, 1995).
The desired iron nanoparticles should be composed of Fe₂O₃ which is one of the most abundant transition metal oxides and ubiquitous in the environment (Bi & Xu, 2012). Toxic effects by these nanoparticles arise from the formation of ROS by converting H₂O₂ or O₂ (Huang et al., 2015). Further studies performed by Wang et al. (2017) regarding the photocatalytic degradation of DNA who found that the majority of intracellular damage especially concerning the DNA arises from ROS.

Reactive oxygen species

As most problems from heavy metals arise from the generation of ROS, the efforts to improve the resistance to their toxic effects need to focus on the repair of the damage of biomolecules and the prevention of further damage. Molecular oxygen itself cannot easily oxidize another molecule due to its spin restriction (Farr & Kogoma, 1991). However, certain cases allow oxidation of molecules by molecular oxygen, e.g. the presence of a paramagnetic center other than that of the molecular oxygen and good catalysts for one-electron reduction such as the transition metals copper and iron. The arising ROS are mostly the superoxide anion (O₂^-), hydrogen peroxide (H₂O₂), hydroxyl radicals (•OH) and singlet oxygen (1O2) (Halliwell, 1991).
O₂^- attacks thiols and ascorbate as well as iron sulfur clusters in proteins and thus disrupts the protein’s structure and leads to a loss of its function. Furthermore, it is able to reduce transition metals such as iron and copper, e.g. Cu(II) to Cu(I). It tends to form the highly reactive hydroperoxyl radical (•OOH) under acidic conditions. The degradation of the superoxide anion happens by spontaneous dismutation into hydrogen peroxide and molecular oxygen (Masoud et al., 2015).
Hydrogen peroxide itself is reactive as well. The exact reaction mechanism in the cell is unknown due to its reaction speed. However, it is known that hydrogen peroxide reacts with reduced copper and/or iron ions to form hydroxyl radicals. As a weak oxidizing agent it attacks thiol groups and reduces glutathione (Liu et al., 2015).
Hydroxyl radicals are highly reactive and react with almost every molecule. Since it is highly reactive, its reaction rate is limited by diffusion. The reactivity of hydroxyl radicals derives from its high standard electrode potential (+2.3 V). Therefore, it is able to oxidize almost any molecule other than ozone (Zhang et al., 2018).
There are several origins for the different types of ROS: Superoxide anion is formed by autoxidation of distinct dehydrogenases and certain reductases, e.g. glutathione reductase. Non-enzymatic production is achieved by autoxidation of certain cellular components such as ubiquinols. Hydrogen peroxidase derives from several oxidases and the superoxide dismutase. UV radiation breaks it down into hydroxyl radicals (Mignolet-Spruyt et al., 2016). Other mechanisms for the generation of hydroxyl radicals are the Fenton and/or Haber-Weiss reaction (Fagali et al., 2015; Kehrer, 2000).
Haber-Weiss: Fe³⁺ + •O₂^- → Fe²⁺ + O₂
Fenton: Fe²⁺ + H₂O₂ → Fe³⁺ + OH^- + •OH
In order to neutralize the ROS and repair the occurred damage bacteria have different mechanisms they can rely on. Two types of superoxide dismutase (SOD) dismutate O2- into H₂O₂: MnSOD and FeSOD (encoded by sodA and sodB) (Broxton & Culotta, 2016). Catalases (encoded by katE and katG) are able to disproportionate H₂O₂ into the nontoxic components H₂O and O₂ (Chaithawiwat et al., 2016). Further detoxification is achieved by the alkyl hydroperoxide reductase Ahp (encoded by ahpC and ahpF) which scavenges various free organic hydroperoxides (Kamariah et al., 2016). These genes are in E. coli under the control of various global regulators such as OxyR and SoxR. The regulators also promote the expression of genes involved in the repair of the damaged cell membranes, DNA and proteins and are activated by a conformational change induced by oxidative stress (Dubbs & Mongkolsuk, 2016; Seo et al., 2015). Furthermore, the tripeptide glutathione plays a vital role in the detoxification of the cell regarding the decomposition of radicals to deal with oxidative stress. The enzymes encoded by gshA and gshB are responsible for the synthesis of glutathione. However, the crucial part in fighting oxidative stress via glutathione is executed by the glutathione peroxidase BtuE, encoded by btuE, and the glutathione reductase GSR, encoded by gor. BtuE uses glutathione as a reducing agent to scavenge hydrogen peroxide and peroxide radicals.
ROOH + 2 GSH → ROH + GSSG + H₂O
GSR then recovers the used glutathione by reducing the generated glutathione disulfide under the consumption of NADPH. The phytochelatin synthase (PCS1) or also called glutathione gamma-glutamylcysteinyltransferase catalyzes the formation of phytochelatin out of the precursor glutathione in plants and fungi (Grill et al., 1989). The enzyme transfers γ-Glu-Cys moieties of glutathione to glutathione or (γ-Glu-Cys)n-Gly, ultimately producing (γ-Glu-Cys)_n+1-Gly peptides with n in the range from 2 to 10 (Figure 4). The phytochelatin synthase plays a crucial role in the metal detoxification process. There are reports (Heiss et al., 2003) that PCS is activated by heavy metal ions such as cadmium and copper in Arabidopsis thaliana cells. We think that heterologous expression of this enzyme in E. coli helps the bacterium to better endure the toxicity of the imported copper ions. The phytochelatin synthase is activated by heavy metal ions such as cadmium which can be seen in figure 4.

Figure 4: A: Schematic illustration of the phytochelatin synthase functioning. The C-terminal domain works as a local sensor of heavy metal ions, such as Cadmium (Cd). Cysteine residues (C) bind Cd ions which are transferred to the activation site in the N-terminal catalytic domain. The activated domain catalyses the relocation of the γ-Glu-Cys moiety of a glutathione (GSH; γ-Glu-Cys-Gly) molecule onto a second glutathione molecule or an existing PCn molecule to form a PC_n+1 product (Cobbett, 1999). B: Catalyzed reaction by phytochelatin synthase. The transpeptidase uses glutathione as a substrate and transfers γ-Glu-Cys moieties on glutathione or (γ-Glu-Cys)n-Gly to form phytochelatins ((γ-Glu-Cys)n-Gly with n between 2 and 10).

In order to increase the cell’s resistance to oxidative stress a sophisticated approach is needed. An overexpression of the oxidative stress dependent regulators SoxR and OxyR should improve the cell’s ability to cope with an elevated number of ROS. Various combinations of the defense mechanisms are tested in order to determine the ideal defense against oxidative stress in order to maximize the longevity in presence of heavy metals. Agents like phytochelatin which are rich in cysteine and/or histidine residues are meant to bind free metal ions to lower the potential for Fenton and/or Haber-Weiss reactions.

The results for the toxicity could be found here

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