4.2.2. Arsenic binding to glutathione

The metabolism of arsenic in the cells involves the reduction of pentavalent arsenic to trivalent arsenic. This reaction consists of a redox cycle involving a bio-thiol (glutathione) with the production of a tris-glutathionyl-arsenite species. The multiple methylations of arsenite by S-adenosylmethionine to the generation of trimethylarsine (hemolytic toxin) also involve glutathione. Glutathione presence in the intermediate conjugate forms of methylated arsenic species helps these molecules to be removed from the cells by the multidrug-resistant proteins (having ATPbinding cassette). Dimethylarsinic acid (carcinogenic end-metabolite) also reacts with glutathione having a high cytolethal effect on cells. Moreover, various enzymes and regulatory elements can contribute to the arsenic biotransformation by contributing individual or multiple cysteine thiol groups in vicinity in proteins, for example, thiol groups required for catalytic activity [41].

#### 4.2.3. Arsenic binding to metallothioneins

Metallothioneins are expressed by various organisms including bacteria, fungi, plants and vertebrates. They belong to a protein family of ubiquitous nature characterized by low molecular weight, high metal and cysteine content. They are capable of binding essential metal ions (zinc, copper) and toxic heavy metals (arsenic, cadmium).

Studies have revealed that bioaccumulation of arsenic in seaweed species Fucus vesiculosus is achieved through the binding of arsenite to the cysteine-rich metallothioneins. Moreover, arsenic is also known to bind to mammalian metallothioneins in rabbit and human species. It is present abundantly in the kidneys and liver of mammals. Further studies on human metallothioneins were consistent with the hypothesis that arsenite has a binding preference for three vicinal thiol groups, with α and β domain of human metallothionein containing 11 and 9 cysteines, respectively. All the 9 cysteines were involved in binding to three arsenite molecules in β domain, while in the case of α domain, only 9 out of 11 cysteine residues were involved in binding to three arsenites. This leaves two cysteine residues protonated with no fourth arsenite engaged in binding [42, 43].

#### 4.2.4. Arsenic binding to ArsD As(III) metallochaperone

Arsenic being the most common toxic element in the environment has resulted in the evolution of arsenic detoxifying mechanisms in nearly all organisms. In archaea and bacteria, trivalent metalloids like arsenite are pumped out of the cell by ArsAB ATPases encoded by various ars operons. Three conserved cysteine residues (Cys12, Cys13 and Cys18) are required for the chaperone activity of ArsD. ArsD also helps to increase the arsenite affinity of Ars A enabling the detoxification of arsenite, even at low concentrations. In the case of ArsA, there are two cysteines (Cys113 and Cys422) in the high affinity metalloid-binding site along with the third cysteine that participates in activation of ATP hydrolysis. In the absence of arsenite, a low basal rate of ATPase activity is shown by ArsA [44].

#### 4.2.5. Arsenic binding to other proteins

4.2. Arsenic binding to specific proteins

Arsenic species are cleared from the blood immediately in humans, but the time of clearance of arsenic from animal species varies noticeably. The retention of arsenic in rat blood is longer when compared with other species. Arsenic has been found to bind to transferrin in hemodialysis patients [39]. Hemoglobin in red blood cells (RBCs) were predicted to be the sites of arsenic accumulation, because hemoglobin constitutes 97% of dry weight of RBCs [40]. The affinity of hemoglobin in rat liver is much higher in rats as compared with humans. The rat and human hemoglobins are tetramers, each consisting of two α-chains and two β-chains. The difference lies in the number of the cysteine residues with rat hemoglobin consisting of three cysteines (Cys111, Cys104 and Cys13) in α-chain, while two cysteines (Cys125 and Cys93) in βchain. On the other hand, human hemoglobin has only one cysteine in α-chain and two

The metabolism of arsenic in the cells involves the reduction of pentavalent arsenic to trivalent arsenic. This reaction consists of a redox cycle involving a bio-thiol (glutathione) with the production of a tris-glutathionyl-arsenite species. The multiple methylations of arsenite by S-adenosylmethionine to the generation of trimethylarsine (hemolytic toxin) also involve glutathione. Glutathione presence in the intermediate conjugate forms of methylated arsenic species helps these molecules to be removed from the cells by the multidrug-resistant proteins (having ATPbinding cassette). Dimethylarsinic acid (carcinogenic end-metabolite) also reacts with glutathione having a high cytolethal effect on cells. Moreover, various enzymes and regulatory elements can contribute to the arsenic biotransformation by contributing individual or multiple cysteine thiol groups in vicinity in proteins, for example, thiol groups required for catalytic activity [41].

Metallothioneins are expressed by various organisms including bacteria, fungi, plants and vertebrates. They belong to a protein family of ubiquitous nature characterized by low molecular weight, high metal and cysteine content. They are capable of binding essential metal ions

Studies have revealed that bioaccumulation of arsenic in seaweed species Fucus vesiculosus is achieved through the binding of arsenite to the cysteine-rich metallothioneins. Moreover, arsenic is also known to bind to mammalian metallothioneins in rabbit and human species. It is present abundantly in the kidneys and liver of mammals. Further studies on human metallothioneins were consistent with the hypothesis that arsenite has a binding preference for three vicinal thiol groups, with α and β domain of human metallothionein containing 11 and 9 cysteines, respectively. All the 9 cysteines were involved in binding to three arsenite molecules in β domain, while in the case of α domain, only 9 out of 11 cysteine residues were involved in binding to three arsenites. This leaves two cysteine residues protonated with no

4.2.1. Arsenic binding to hemoglobin

66 Arsenic - Analytical and Toxicological Studies

cysteines in β-chain [3].

4.2.2. Arsenic binding to glutathione

4.2.3. Arsenic binding to metallothioneins

fourth arsenite engaged in binding [42, 43].

(zinc, copper) and toxic heavy metals (arsenic, cadmium).

Trivalent arsenic species are also known to bind to other proteins like actin, tubulin, estrogen receptor and glucocorticoid receptors. Arsenite can bind to Kelch-like ECH-associated protein 1 (KEAP 1). This is a major antioxidant-sensing protein which acts at low Kd values. One of the most common motifs present in many proteins consists of two cysteine residues separated by two amino acids (CXXC). The presence of cysteine residues increases with increasing complexity of the organisms, making humans vulnerable to arsenic toxicity because of the high affinity of arsenic for cysteine residues.

One such important cell surface protein consisting of highly conserved cysteine residues is connexin 43 (Cx43)—a widely expressed gap junctional protein important for cell death, proliferation and differentiation [45]. Cx43 has nine Cys residues, six of which are in the extracellular domain and three in the intracellular domain. Six connexin monomers form a hemichannel called connexin. In the plasma membrane, one connexin can dock to another connexin in the plasma membrane of an adjacent cell resulting in the formation of complete gap junction channel. A hemichannel formed by single type of connexin isoforms is called homomeric hemichannel or consists of multiple types of isoforms called heteromeric hemichannel. Two identical homomeric or heteromeric hemichannels dock to form a homotypic channel, and two different homomeric or heteromeric hemichannels dock to form a heterotypic channel (Figure 6). Recent in silico studies (Hussain et al., manuscript communicated) in combination with cellular and biochemical analysis revealed insights into the binding modes of arsenite to conserved Cys groups in Cx43. In Cx43, As+3 can be bound to three cysteines in the intracellular domain in a monovalent fashion as they are free cys, while it can bind the extracellular domain cysteines in either monovalent, divalent or trivalent fashion depending on the state and location of the protein in the cell. Arsenite ion (As+3) can attack the free sulfhydryl group until all the valencies of the As+3 are satisfied by covalent bonding to the sulfur from the cys residues. This profoundly affects the Cx43 primary, secondary, tertiary and the quaternary structure. This study is the first of its kind which shows that arsenic can directly bind to Cx43 via its highly conserved cysteine residues causing misfolding of Cx43, which leads to alteration of transportation, localization and oligomerization of Cx43. Further experiments revealed that Cx43 was colocalizing with ER marker (calnexin), revealing the inability of Cx43 to be transported beyond endoplasmic reticulum/endoplasmic reticulum

Figure 6. Hierarchy of structures involved in the formation of gap junction intercellular communication (GJIC).

Golgi intermediate compartment (ER/ERGIC) (Hussain et al., manuscript communicated). This loss of Cx43 composed of functional gap junctions on the cell surface has deleterious effect on cellular homeostasis (Figure 7).

Another such important cellular factor involved in cellular stress is DJ-1. DJ-1 is a 20KDa, homodimeric protein containing a nucleophilic elbow forming the active site of the protein. There are three important and conserved cysteine residues (Cys46, Cys53 and Cys106) in the DJ-1 protein, of which Cys53 and Cys106 are exposed. Cys106 has been found to be a prominent player in the nucleophilic groove that binds to divalent ions like zinc (II), copper (II) [48] and mercury [49] in vitro. Interaction with metal ions might be a possible mechanism of DJ-1 mediated cellular protection against metal-induced toxicity. Arsenic in the form of arsenite (As (III)) has been found to interact with three thiol group of cysteine residues [3]. Therefore, there is a possibility that arsenic binds to the nucleophilic groove in the homodimer of DJ-1. Oxidation state of the Cys106 is one of the determining factors behind the activity of the protein. Cysteine has the propensity to bind to three oxygen atoms resulting in the formation of the three forms—SOH, SO2H and SO3H. The presence of the SOH and SO2H form activates the protein causing its translocation into the nucleus. Upon activation, DJ-1 regulates the activity of several transcription factors like nuclear factor erythroid 2-related factor 2 (Nrf2), polypyrimidine tract-binding protein-associated splicing factor (PSF) and sterol regulatory element-binding protein (SREBP), signal transducer and activator of transcription 1 (STAT1) and Ras-responsive element-binding protein (RREB1). DJ-1 has been found to inhibit phosphatase and tensin homolog (PTEN), an inhibitor of the AKT (protein kinase B) signaling pathway, resulting in enhanced cell proliferation. DJ-1 also functions in the sequestration of the death domain-associated protein (DAXX) in the nucleus. DAXX is required in the cytoplasm for

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Figure 7. Arsenic binding causes alteration in trafficking of connexin 43 to the cell membrane.

Arsenic is considered a group 1 carcinogen by the International Agency for Research on Cancer (IARC) and causes cancers of the lung, liver and skin [46]. Gap junction intercellular communication has been found disrupted in many tumors and malignancies. Gap junctions are considered tumor suppressors, and the persistent downregulation of gap junction proteins makes cells susceptible to cancer [47]. Decreased or diminished expression and/or function of Cxs has been observed in most tumor cell lines and in solid tissue tumors, including melanomas. Our study revealed that arsenic causes disruption of gap junction intercellular communication both in vivo and in vitro. Arsenic is considered a weak mutagen; therefore, recent trends in the field have focused on deciphering the role of non-mutagenic pathways like cell–cell communication in arsenic-induced cancer. Our study revealed that arsenic induces disruption of gap junctions which are considered as tumor suppressors, thereby putting forward new nonmutagenic pathways which may be altered during the course of arsenic-induced carcinogenesis. Mechanisms of Arsenic-Induced Toxicity with Special Emphasis on Arsenic-Binding Proteins http://dx.doi.org/10.5772/intechopen.74758 69

Figure 7. Arsenic binding causes alteration in trafficking of connexin 43 to the cell membrane.

Golgi intermediate compartment (ER/ERGIC) (Hussain et al., manuscript communicated). This loss of Cx43 composed of functional gap junctions on the cell surface has deleterious effect on

Figure 6. Hierarchy of structures involved in the formation of gap junction intercellular communication (GJIC).

Arsenic is considered a group 1 carcinogen by the International Agency for Research on Cancer (IARC) and causes cancers of the lung, liver and skin [46]. Gap junction intercellular communication has been found disrupted in many tumors and malignancies. Gap junctions are considered tumor suppressors, and the persistent downregulation of gap junction proteins makes cells susceptible to cancer [47]. Decreased or diminished expression and/or function of Cxs has been observed in most tumor cell lines and in solid tissue tumors, including melanomas. Our study revealed that arsenic causes disruption of gap junction intercellular communication both in vivo and in vitro. Arsenic is considered a weak mutagen; therefore, recent trends in the field have focused on deciphering the role of non-mutagenic pathways like cell–cell communication in arsenic-induced cancer. Our study revealed that arsenic induces disruption of gap junctions which are considered as tumor suppressors, thereby putting forward new nonmutagenic pathways which may be altered during the course of arsenic-induced carcinogenesis.

cellular homeostasis (Figure 7).

68 Arsenic - Analytical and Toxicological Studies

Another such important cellular factor involved in cellular stress is DJ-1. DJ-1 is a 20KDa, homodimeric protein containing a nucleophilic elbow forming the active site of the protein. There are three important and conserved cysteine residues (Cys46, Cys53 and Cys106) in the DJ-1 protein, of which Cys53 and Cys106 are exposed. Cys106 has been found to be a prominent player in the nucleophilic groove that binds to divalent ions like zinc (II), copper (II) [48] and mercury [49] in vitro. Interaction with metal ions might be a possible mechanism of DJ-1 mediated cellular protection against metal-induced toxicity. Arsenic in the form of arsenite (As (III)) has been found to interact with three thiol group of cysteine residues [3]. Therefore, there is a possibility that arsenic binds to the nucleophilic groove in the homodimer of DJ-1. Oxidation state of the Cys106 is one of the determining factors behind the activity of the protein. Cysteine has the propensity to bind to three oxygen atoms resulting in the formation of the three forms—SOH, SO2H and SO3H. The presence of the SOH and SO2H form activates the protein causing its translocation into the nucleus. Upon activation, DJ-1 regulates the activity of several transcription factors like nuclear factor erythroid 2-related factor 2 (Nrf2), polypyrimidine tract-binding protein-associated splicing factor (PSF) and sterol regulatory element-binding protein (SREBP), signal transducer and activator of transcription 1 (STAT1) and Ras-responsive element-binding protein (RREB1). DJ-1 has been found to inhibit phosphatase and tensin homolog (PTEN), an inhibitor of the AKT (protein kinase B) signaling pathway, resulting in enhanced cell proliferation. DJ-1 also functions in the sequestration of the death domain-associated protein (DAXX) in the nucleus. DAXX is required in the cytoplasm for providing the second activation signal to the phosphorylated apoptosis signal-regulating kinase 1 (ASK1) protein, which then triggers the apoptotic pathway. As a result, unavailability of DAXX in the cytoplasm hinders the initiation of the apoptotic pathway. Under condition of excess oxidative stress, the SO3H form prevails which inactivates the protein and retranslocates back to the cytoplasm. As a result, the entire antioxidant response regulated by the activated DJ-1 protein is inhibited. Moreover, DAXX protein also becomes free, which then translocates into the cytoplasm and provides the required second activation signal to the phosphorylated ASK1 protein.

the detection process. Except optical (fluorescence and UV–Vis) detection methods, other available methods need complex experimental setup; hence, they are far from 'on-field' application purpose. Simplicity, low-cost and 'on-field' application possibilities make optical

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Optical sensors can be of different types depending upon the material used for sensing. The first one is nanomaterial-based assays for the detection of the arsenic in different mediums. Though the detection of arsenic is tough, but researchers are able to draw an outline about the ligands which can bind arsenic, and these ligands can be used as a binding unit in a sensing material which leads to either color change or change in emission spectrum. As arsenic is very much labile towards thiol group, a bunch of thiolated ligands are reported for arsenic binding. These ligands are dithiothreitol (DTT), reduced glutathione (GSH) and cysteine, and Figure 8 describes the chemical structure of these three ligands. Arsenic can bind with GSH and cysteine by forming As-O bond also, if no free –SH available. Except thiolated ligands, there are some ligands like humic acid [60] and N-(dithiocarboxy)-N-methyl-D-glucamine [61] which can also bind As(III) by forming As-O bond. Keeping this information in mind, gold nanoparticle-based sensors were reported for As(III) detection. The surface of the gold nanoparticles can be modified by the thiolated ligands, which after binding with As(III) showed a drastic color change to indicate the presence of the toxicant in the aqueous medium [62]. Aptamer-conjugated nanoparticles are also very effective composites which can detect arsenic in aqueous medium [63, 64] by changing the color. In all these types of detection assays, aggregation of the nanoparticles is the predominant factor to show the color change. Though these kinds of materials are responsive towards arsenic, but sensitivity is one of the

Both selectivity and sensitivity are important for effective detection of arsenic. Small molecules are developed to detect different forms of arsenic in aqueous medium having good selectivity over other toxicants as well as good sensitivity. Baglan M et al. have reported a cysteine-fused tetraphenylethene, which can bind with As3+, and showed aggregation-induced emission as a signal [65]. Here, also the thiol group of cysteine acts as the dominating factor for As3+ binding and leading to the close proximity arrangement of the tetraphenylethene. More toxic As3+ can be distinguished over less toxic As5+ using this system, and the detection limit tends to 0.5 ppb, which is lower than the limit according to the World Health Organization (WHO) [66]. Keeping besides the thiol systems, Somentah et al. have designed a simple Schiff base system which

most exciting and effective for the detection of pollutants. In this work they have designed a molecule which is initially not showing any fluorescence emission, but after selective addition

chelation-enhanced fluorescence (CHEF) [67]. Development of arsenic sensor is evolving year

<sup>3</sup> fluorescence, signal is turned on due to intermolecular H-bonding leading to

<sup>3</sup> fluorometrically. 'Off–on' system in fluorescence is always

sensing technique versatile.

issues which prevent these from field effectiveness.

Figure 8. Chemical structures of thiolated ligands (DTT, GSH and cysteine).

can identify the most toxic AsO3

of AsO3
