Effect of UV radiation on the expression of bioactive molecules in human skin cells
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\r\n\tEditors hope to build a line of transformative research based on the adaptation of energy and design solutions from natural models to technical models, using methodologies and solutions which will open new areas of work about how to solve efficiency requirements based on natural solutions honed by evolution. This process is based on creativity and forces researchers to think out of their boxes and open new scientific challenges. What drives the research is the urge to find alternative, more efficient solutions to tackle problems. Due to the wide range of possibilities offered by biodiversity’s tested solutions, the methodology prioritize (although not exclude) solution-based approaches.
Skin cancer represents a major, and growing, public health problem, and is the most common type of cancer observed in Caucasians [1-3]. The three most common forms of skin cancer are basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and melanoma. BCC and SCC are together known as non-melanoma skin cancers (NMSC), and are both derived from keratinocytes whereas melanomas are derived from melanocytes [3-6]. SCCs can undergo metastasis; BCCs rarely do, while melanomas can be highly metastatic [5, 6].
The ultraviolet (UV) radiation component of sunlight is acknowledged to be the main carcinogen implicated in the formation of skin cancer. UV radiation can be divided into three components: UVC (100-280 nm), UVB (280-320 nm) and UVA (320-400 nm). Ozone depletion, seasonal and weather variations affect the amount of UV radiation reaching the Earth’s surface [7]. UVC and most of the UVB radiation emitted from the sun is blocked from reaching the Earth’s surface by the ozone layer. The component of UV light that reaches the Earth’s surface consists of 90-95% UVA and 5-10% UVB [3, 8]. The penetration of shorter-wavelength UVB radiation is predominantly confined to the epidermis while UVA penetrates into the dermis because of its longer wavelength [9].
UVB can cause sunburn, inflammation, DNA mutations and membrane damage as well as skin cancer [8, 10, 11]. It is known that UVB directly damages DNA and can induce Reactive Oxygen Species (ROS) by interactions with chromophores in the skin [12]. The DNA damage caused by UVB irradiation typically results in the formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) photoproducts. The mutations are frequently found in the p53, p16, PTCH and INK4α/CDKN2A genes of skin cancer patients [13, 14]. Inflammation plays a significant role in creating an environment where cells possessing mutated DNA can become carcinogenic. UVA can cause premature skin ageing, wrinkle formation, blotching and induces sunburn cell formation in the epidermis, as well as skin cancer [8, 10, 15]. It affects keratinocytes at a transcriptional level by altering the expression of genes involved in apoptosis, cell cycle, DNA repair, signal transduction, RNA processing and translation, and metabolism [9]. UVA can cause DNA damage by generating ROS [12] resulting in genomic damage e.g. single-stranded breaks, protein-DNA crosslinks, and oxidative base damage (i.e. 8-oxo-7,8-dihydroxyguanine) [16]. It can also initiate signal transduction pathways [13, 17] as well as inducing the expression of cytokines such as Interleukin (IL)-6, heme oxygenase-1, and cyclo-oxygenase [18] as well as inflammatory mediators such as tumor necrosis factor-α (TNFα) [15, 19].
While UVB has been thought to be the main contributor toward skin cancers, based largely on the DNA action spectrum of UV radiation, UVA has more recently been acknowledged as playing an important role in this process [9, 11, 20]. While UVA does not produce an inflammatory response like that of UVB, it produces ROS and as such activates many of the same signalling pathways [13]. It is clear that doses of UVB, UVA and solar stimulated UV that are too low to cause inflammation can induce mutations in epidermal cells. However, this does not exclude a role for ROS from inflammatory cells contributing to skin carcinogenesis, but it may be important for tumour progression [20]. UV-induced inflammation seen in the skin involves the action of many molecules. Of these inflammatory molecules TNFα plays a major role in UV-irradiated inflammation in the skin [15, 19]. TNFα is cleaved from its membrane-bound precursor by the action of the metalloprotease, Tumour Necrosis Factor-α Converting Enzyme (TACE) [21, 22]. While UVB radiation increases the release of TNFα from skin cells, it is not known whether this is due to increased TACE activity and/or expression. However, before TACE is activated it is cleaved from its proform by the action of furin, a proprotein convertase [23, 24]. Furin can cleave other proteases such as matrix metalloproteases (MMPs) [23, 24]. Exposure to UVB radiation also increases MMP activity in skin cells [25]. While furin is expressed in skin cells, the effect UV radiation has on its expression and/or activity and that of the proteases it activates is not fully known. As a result of elevated furin levels in a mutated cell, enhanced TACE activity would see an increase in the secretion of TNFα thereby sustaining a localised inflammatory environment allowing for the development of carcinogenic cells. As furin activates MMP activity, these carcinogenic cells have the potential to become metastatic. This review investigates the role that furin plays in the activation of TACE and MMPs and the effect that this has on a skin cells exposed to UV radiation, as well as that its role in cancer cells which undergoes metastasis, and how an understanding of the role played by this proprotein convertase, may assist in the design of new inhibitors which have therapeutic potential.
High doses of UV can induce inflammation in the skin that results in the appearance of macrophages and other leucocytes [15, 26]. Along with the activation of these cells, many mediators of inflammation are also seen including; prostaglandins [18], nitric oxide (NO) [27] and ROS [12, 17], and cytokines such as interleukin (IL)-1, interferon (IFN)-γ, and TNFα [14, 19, 28, 29]. ROS can cause DNA strand breaks as well as lipid peroxidation, membrane and protein damage [12, 30]. The effects of UV radiation on the levels of these proinflammatory molecules in skin cells are seen in Table 1 [8, 15, 31-33].
Inflammatory mediators such as IL-1, TNFα and IL-6 have been postulated to play a major role in both melanoma [34] and NMSC formation [26, 35, 36]. Male mice are known to be more sensitive to UVB-induced skin carcinogenesis than female mice [4], which is consistent with human studies showing men having a higher incidence of skin cancer than women [20]. Damian et al. [20] found that while women developed a larger inflammatory response to UVB radiation, men had lower antioxidant levels in the skin resulting in a higher level of oxidative damage to DNA, and were more sensitive to UV immunosuppression. This suggests that UV-induced immunosuppression and DNA damage plays a greater role in the formation of skin cancers in men compared to women [20]. IL-1α and IL-1β are both induced in keratinocytes exposed to UVB radiation [31, 37]. IL-1α has been shown to enhance the expression and release of TNFα from UVB-irradiated keratinocytes [38, 39], while IL-1β enhances the expression of matrix metalloprotease (MMP)-9 in these irradiated cells [40]. Apart from IL-1β, UVB can stimulate MMP-9 expression in human skin via the induction of Activator protein-1 (AP-1) and NFκB activities [41].
\n\t\t\t\tMediator\n\t\t\t | \n\t\t\t\n\t\t\t\tProduced By\n\t\t\t | \n\t\t\t\n\t\t\t\tFunction\n\t\t\t | \n\t\t\t\n\t\t\t\tReferences\n\t\t\t | \n\t\t
TNFα | \n\t\t\tKeratinocytes Mast cells Dermal fibroblasts Langerhans cells | \n\t\t\tLangerhans cell migration, sunburn cell information, stimulates prostaglandin (PG) synthesis, changes in adhesion molecule expression | \n\t\t\t[15, 31, 33] | \n\t\t
IL-1α | \n\t\t\tKeratinocytes Langerhans cells | \n\t\t\tSimulates PG synthesis, increases TNFα and IL-6, inhibited by IL-1 receptor antagonist | \n\t\t\t[15, 33] | \n\t\t
IL-1β | \n\t\t\tKeratinocytes Langerhans cells | \n\t\t\tLangerhans cell migration | \n\t\t\t[15, 31, 33] | \n\t\t
IL-6 | \n\t\t\tKeratinocytes, Langerhans cells | \n\t\t\tFever Severe sunburn | \n\t\t\t[8, 15, 33] | \n\t\t
IL-10 | \n\t\t\tMacrophages Melanocytes | \n\t\t\tBlocks cytokine production by T cells, macrophages and NK cells Decreases antigen presentation, Increases IL-1 receptor antagonist | \n\t\t\t[33] [8, 15, 32, 33] | \n\t\t
IL-I2p40 (not bioactive) | \n\t\t\tKeratinocytes, Dendritic cell, Langerhans cells | \n\t\t\tDecreases Th1 response Decreases antigen presentation | \n\t\t\t[33] | \n\t\t
IFNγ | \n\t\t\tT cells | \n\t\t\tTriggers apoptosis T-cell mediated tumour cell destruction | \n\t\t\t[33] | \n\t\t
PGE2\n\t\t\t | \n\t\t\tKeratinocytes, Mast cells | \n\t\t\tErythema Decreases antigen presentation Increases IL-4, decreases IL-12 | \n\t\t\t[15, 33] | \n\t\t
Histamine | \n\t\t\tMast Cells | \n\t\t\tIncreases release of PG Inhibit lymphocyte functions like IL-2 and IFNγ | \n\t\t\t[8, 15, 33] | \n\t\t
Effect of UV radiation on the expression of bioactive molecules in human skin cells
UVB radiation can increase cyclo-oxygenase (COX)-2 expression and activity in keratinocytes [20, 28, 42]. High levels of COX-2 activity have been observed in human epithelial skin cancers [43]. Nonsteroidal anti-inflammatory drugs can inhibit COX-2 activity and subsequent PGE formation in the skin, and have been used in the treatment of actinic keratosis (AK) [44], BCC, SCC and melanoma [45]. This suggests a role for COX-2 in the formation of skin cancers, and high levels of activity have been observed in many of these tumours [46].
Upregulation of TNFα is a key early response observed in keratinocytes exposed to UVB radiation [8, 38, 47] and represents an important component of the inflammatory cascade in skin. The expression of TNFα mRNA was enhanced a few hours post-UVB irradiation in both keratinocytes and dermal fibroblasts [38, 47]. IL-1α was shown to stimulate TNFα expression in UVB-irradiated keratinocytes [47] and melanocytes [48]. While Bashir et al. [38] observed that TNFα expression in keratinocytes was only induced by UVB irradiation, others have shown that UVA can also induce expression in these cells [49, 50]. This increase in TNFα released by the cells is due to elevated gene transcription [38, 49]. The IL-1α formed in the skin, can in turn, induce mast cells to express inflammatory cytokines (e.g. TNFα and IL-1α), as well as prostaglandins which can enhance the inflammation caused by direct UV exposure on the epidermis [15, 20, 28, 51]. Histamine released from the mast cells can induce vasodilation of the surrounding blood vessels, which assists leucocytes in undergoing diapedesis and entering this region [20, 51]. UVB radiation can induce the synthesis and release of IL-6 and IL-8 from irradiated keratinocytes and fibroblasts [33, 36, 37, 51]. IL-8 assists in the homing of leucocytes, primarily neutrophils, from surrounding blood vessels into the inflamed region, while IL-6 can trigger the activation of monocytes and other infiltrating leucocytes to secrete cytokines and chemokines [51]. Figure 1 shows the complex interaction that occurs between different bioactive molecules in the skin following exposure to UV radiation.
The inflammatory response seen in the skin following exposure to UV radiation. Inflammation can be induced as a direct result of UV exposure on epidermal cells, or due to the release of secreted molecules, which in turn induce the release of inflammatory mediators from the dermis, as well as attracting inflammatory cells from circulation into this region of the skin. The infiltrating monocytes and macrophages, which enter the irradiated skin tissue in turn, secrete mediators that prolong the inflammatory response. See text for details and references.
TNFα can induce the expression of adhesion molecules and chemokines in surrounding epithelial cells, resulting in the recruitment of inflammatory leucocytes from surrounding blood vessels via diapedesis [15, 20, 51, 52]. These inflammatory cells in turn can express additional cytokines that form a positive feedback loop that further upregulates TNFα as well as downstream TNF
TNFα plays a pro-inflammatory role in the skin due to; (a) the direct effects of UV radiation and (b) the indirect effects of inflammatory cells that chemotax to the skin. UV- and inflammatory cell-derived cytokines further enhance TNFα gene transcription in human skin cells [38], which can again increase its production by epidermal cells. In contrast, clustering and internalization of the TNF receptors may lessen the cell’s response to TNFα, which may account for why the upregulation of TNFα mRNA is not sustained over time in culture [20]. For further information on the complex interplay of cytokines, chemokines and other mediators in UV-induced inflammation please refer to the following reviews [15, 20, 41, 42].
TNFα, is a member of the TNF ligand superfamily, and is a type II transmembrane glycoprotein of 234 amino acids possessing an extracellular carboxy-terminus and a cytoplasmic amino group [53, 55, 56]. It can exist in one of two forms; a 26 kDa membrane-bound form (mTNFα) and a 17 kDa soluble form (sTNFα). sTNFα is cleaved from its membrane bound precursor between Ala76 - Val77 by the action of the metalloprotease TACE [22, 55].
Numerous cells produce TNFα, including macrophages, leucocytes, dendritic cells, keratinocytes, melanocytes and fibroblasts [8, 47, 57, 58]. It plays a role in apoptosis, cellular proliferation, differentiation, inflammation, tumorigenesis, viral replication, immune response to extracellular stimuli, as well as in local and systemic inflammation [21, 53, 55-57, 59]. Most of the cellular actions described for TNFα correspond to its secreted, mature soluble form. There is increasing evidence that mTNFα is also biologically active [58]. Both forms of TNFα can specifically bind to one of two receptors: TNF-R1 (CD120a receptor), a 55 kDa protein; TNF-R2 (CD120b receptor), a 75 kDa protein [57]. The receptors are both transmembrane glycoproteins, and display a high degree of structural homology and are expressed on most cell types [60].
TNF-R1 is expressed on a wide range of cell types and its signalling mediates cytotoxicity, cell proliferation, antiviral activity and many of the proinflammatory actions of TNFα [58, 61]. TNF-R2 is expressed on a limited range of cells, including leucocytes, endothelial cells, Langerhans cells (LC) and epithelial cells but its actions are less clear [58, 61]. Membrane-bound TNF-R1 and TNF-R2 can be cleaved by TACE to release the soluble forms of these receptors and this process is activated by IL-10 [58]. The soluble forms of TNF-R may act as (a) an antagonist to the surface receptors by competing for sTNFα or (b) an agonist by stabilizing the TNF trimer; therefore maintaining saturating concentrations in extracellular fluids [58, 62].
When TNFα is bound to the TNF-R1 receptor it plays a role in UVB-induced apoptosis in keratinocytes [54, 63]. Transgenic mice deficient for either TNF-R1 and/or TNF-R2 have been shown to be less susceptible to UVB-induced skin tumours than were wild type controls [64]. Through the use of TNF-R1 [65, 66] and TNF-R2 [65] gene-targeted mutant mice, it has been shown that TNF-R1 plays a decisive role in the host’s defence against microorganisms, while TNF-R2 plays a role in the induction of tissue necrosis. Through the use of agonist and antagonist antibodies, TNF-RI was shown to be the main mediator of TNFα action in the cell [67].
Dermal injection of TNFα resulted in the accumulation of dendritic cells in draining lymph nodes as well as in impairment of contact hypersensitivity (CHS) in the skin [60, 68]. This suggests that TNFα induces the migration of LC from the skin to the surrounding regional lymph nodes. Streilein and colleagues [69, 70] observed that UVB indirectly induced TNFα, which then caused morphologic and functional changes on LC resulting in the impairment of CHS, suggesting that TNFα plays a role in this process.
Studies using TNF-R1(-) mutant mice have shown that TNFα was not involved in UVB-induced immunosuppression [71]. UVB-induced immunosuppression is implicated in the pathogenesis of skin cancers, and is mediated in part by cis-urocanic acid (cis-UCA) [72, 73]. trans-Urocanic acid, a deamination product of histidine, is a major chromophore present at high concentrations in the stratum corneum [73]. Upon exposure to UV radiation, trans-UCA undergoes a photoisomerization to its cis-isomer until equilibrium is reached. In humans, this occurs after one minimal erythemal dose of UV radiation, which is the lowest dose that can induce a visibly perceptible erythema [72, 73]. cis-UCA does not exert its immunosuppressive effects via TNFα, but through other factors such as prostaglandin E2 [72]. Amerio et al. [71] showed that in TNF-R1 and TNF-R2 double knockout mice, TNFα played a minimal role in UVB-induced immunosuppression and therefore cannot be considered as a major mediator of cis-UCA-induced immunosuppression. While TNFα does not play a major role in UV-induced immunosuppression [60, 71] it does play a significant role in UV-induced inflammation [20] as well as in other inflammatory diseases such as rheumatoid arthritis, psoriasis, systemic lupus erythematosus and cancer [21, 38, 46, 74].
TNFα is cleaved from its proform by the action of the metalloprotease TACE [75]. This enzyme is a member of the disintegrin and metalloprotease (ADAM) family of proteases, and is also known as ADAM 17 [22, 75-77]. ADAM proteases belong to the adamalysin/reprolysin subfamily of the metzincin superfamily, and contain a Zn2+-dependent catalytic domain [75, 77].
TACE was first purified, characterized and cloned in 1997 and is a multi-domain type I transmembrane protein of 824 amino acids in length [22, 76, 78]. While its amino acid sequence shows relatively low homology to other ADAM family members, its structure contains all the domain regions, which are characteristic for this family of metalloproteases [22, 76, 79]. Structurally TACE consists of a signal peptide followed by a pro, catalytic, disintegrin, cysteine-rich, transmembrane and cytoplasmic domain [55, 80]. The catalytic domain contains the zinc-binding consensus motif HEXGHXXGXXHD involved in coordinating Zn2+ with His residues and creating the active site of the enzyme [79, 81]. The cysteine-rich domain may play a role in enzyme maturation or substrate recognition [75, 76].
TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. In order for TACE to be activated its prodomain is removed at the furin cleavage site RVKR (Arg-Val-Lys-Arg) localized between the pro- and the catalytic domain, and is due to the action of a furin-type proprotein convertase [24, 77, 82-84]. In mammalian cells, proTACE is located in the endoplasmic reticulum and the proximal Golgi body whereas the mature form is located both intracellularly and on the cell membrane [83, 85]. TACE maturation is closely linked to the transport of proTACE through the medial Golgi, where upon exit, prodomain removal occurs before the enzyme reaches the cell’s surface [77].
Apart from TNFα, TACE cleaves a wide range of molecules including transforming growth factor α (TGFα), amphiregulin, neuregulin, growth hormone receptor, TNF-R1, TNF-R2, L-selectin, amyloid precursor protein and IL-6R [77, 86-89]. TACE-knockout mice are far less efficient at processing TNFα on the cell membrane compared to wild type controls [75, 86]. This suggests that TACE is the main protease responsible for the processing of TNFα in the cell. Although some matrix metalloproteases (MMP) can cleave TNFα, the cleaved products are inactive due to hydrolysis occurring at different sites within the molecule [75, 81, 89].
Some metalloproteases are activated in epidermal cells following UV radiation [90-93]. Piva and co-workers found that there were a number of proteases whose activity was upregulated in UVC- or UVB-irradiated HeLa cells [91-93]. These enzymes included aminopeptidases and a “TGFαase” [91, 92]. On re-evaluation of their data, the TGFαase in questions is most likely TACE, because (a) the later enzyme is known to cleave TGFα among other growth factors [81, 88] and (b) the substrate used in these studies was a nonapeptide based on the N-terminal cleavage site of TGFα [90-93]. In cells undergoing UV-induced apoptosis, the level of cell surface protease activity (aminopeptidase and “TGFαase”) was shown to be higher than that seen in viable or necrotic cells [91, 93]. The results of these studies were the first to show that TACE activity was elevated in cells exposed to UV radiation. Recently Skiba et al. [29] reported that UVA and UVB irradiation increased TACE mRNA levels in HaCaT cells, with higher induction induced by UVA. The expression patterns for both UVA- and UVB-irradiated cells in general appeared to be constant, although mRNA levels were significantly higher than controls throughout the 48 h post-exposure period [29].
In UV-irradiated HaCaT cells, TACE was responsible for the increased cleavage of EGF family members [28, 94]. Inhibition of TACE by metalloprotease inhibitors reduced the release of these growth factors, resulting in an increase in apoptotic cell death [28, 94]. It appears that TACE mediates a EGF receptor/AKT signalling pathway in these cells that is activated as a result of its cleavage of EGF family members. In HaCaT cells exposed to UVA-radiation TACE mediated EGF receptor activation and cell cycle progression, which suggests that UVA, at non-lethal doses, has the potential to be a skin cancer promoter [28, 94]. TACE has also shown to be overexpressed in some tumours [21, 46, 56], as well in a large number of skin cancer cells lines compared to their non tumorous counterparts [28, 94]. It is also known that members of the EGF family are overexpressed in skin cancers [95], and this could be a mechanism by which skin cancer growth is stimulated by autogenic growth factors. The results of these recent studies suggest that inhibition of TACE following UV radiation may prevent the stimulation of surviving irradiated cells. This has the potential in reducing the incidence of skin cancer that may arise from prolonged sun exposure. It is not clear if the increase in TACE activity seen in UV-irradiated skin cells is due to increased numbers or a higher level of activity. Furin is known to activate TACE [83, 85, 96] as well as matrix metalloproteases (MMP) [97, 98] and may indirectly play a role in this process.
Furin, also known as PACE, is a 94 kDa, type I transmembrane, Ca2+-dependant serine protease. It is a member of the proprotein convertase (PC) family which is related to the bacterial subtisilin enzyme [23, 97-99]. The PC family consists of seven distinct members (furin and PC1-PC7) that vary in regards to their tissue and subcellular distribution as well as enzymatic and biochemical properties [23, 24, 97, 100]. Furin, PACE 4, PC5/6 and PC7/8 are widely expressed in the epidermis whereas PC2 and PC1/3 are limited to neuroendocrine tissues and PC4 is restricted to the testis [23, 24, 98]. The PC enzymes recognize basic motifs, cleaving after paired basic residues (PC2 and PC1/3); or after a canonical Rx (R/K) R (Arg-x-(Arg/Lys)-Arg) motif (furin and PACE4) [24, 97, 98, 100-102]. Both PC7 and furin share cleave similar substrates and the selectivity of which depends on their cellular localization. As their cytosolic domains regulate intracellular trafficking it is likely that the cellular localization of PC7 differs to that for furin [85].
Structurally furin and other PCs consist of a signal peptide followed by pro, catalytic, middle, and cytoplasmic domains, respectively [24]. The signal peptide directs the translocation of the peptide chain to the endoplasmic reticulum and the secretory pathway [82, 97, 103]. The pro-region is cleaved in the endoplasmic reticulum, where it then associates with the catalytic domain and helps to guide the protein through this region to the Golgi apparatus where it becomes catalytically active [97, 103]. The trans-membrane region anchors the enzyme in the membrane of the trans Golgi network (TGN) or on the cell membrane. The cytosolic tail contains the information necessary for furin’s sorting to various intracellular compartments [82, 97, 103]. In the epidermis, furin can exist either as: (a) a mature 97 kDa membrane bound enzyme or (b) a smaller 75 kDa form that lacks the transmembrane domain [97]. This suggests that post-translational cleavage at the C-terminus occurs within in the cell [97, 98, 103]. Furin and other PC family members process inactive precursor proteins to their functional or mature form, and these include growth factor receptors, growth factors, hormones, plasma proteins, and MMPs [23, 24, 97, 98, 103] as seen in Table 2. PC family members play crucial roles in a variety of physiological processes and are involved in the pathology of diseases such as cancer and viral infection [23, 101, 103-106].
\n\t\t\t\tFunctional group\n\t\t\t | \n\t\t\t\n\t\t\t\tSubstrate\n\t\t\t | \n\t\t\t\n\t\t\t\tReferences\n\t\t\t | \n\t\t
Serum proteins | \n\t\t\tVon Willebrand Factor | \n\t\t\t[107] | \n\t\t
\n\t\t\t | Coagulation factor IX | \n\t\t\t[108] | \n\t\t
Signalling peptides | \n\t\t\tEndothelin-1 | \n\t\t\t[103, 109] | \n\t\t
Growth factors | \n\t\t\tTGFβ | \n\t\t\t[103, 110] | \n\t\t
\n\t\t\t | Vascular endothelial growth factor (VEGF) | \n\t\t\t[111] | \n\t\t
\n\t\t\t | β-Nerve growth factor | \n\t\t\t[112] | \n\t\t
Membrane proteins | \n\t\t\tMT1-MMP | \n\t\t\t[86, 113, 114] | \n\t\t
\n\t\t\t | TACE | \n\t\t\t[77, 99, 115] | \n\t\t
Transmembrane receptors | \n\t\t\tNotch1 Receptor | \n\t\t\t[98, 116] | \n\t\t
\n\t\t\t | Insulin growth factor 1 receptor | \n\t\t\t[117] | \n\t\t
Extracellular matrix proteins | \n\t\t\tN-Cadherin | \n\t\t\t[113, 118] | \n\t\t
\n\t\t\t | Integrin α-chain subunits | \n\t\t\t[119] | \n\t\t
Viral proteins | \n\t\t\tEbola virus glycoprotein | \n\t\t\t[103, 120] | \n\t\t
\n\t\t\t | Papillomavirus minor capsid protein L2 | \n\t\t\t[121] | \n\t\t
Bacterial toxins | \n\t\t\tAnthrax toxin | \n\t\t\t[122] | \n\t\t
\n\t\t\t | \n\t\t\t\tClostridium septicum alpha-toxin | \n\t\t\t[103, 123] | \n\t\t
Some biological molecules cleaved by furin
As a result of the role furin plays in many disease states, considerable effort has been directed at designing specific inhibitors that may have therapeutic applications. The first furin inhibitors that were synthesised where peptidyl chloromethyl ketones [124]. The next major furin inhibitor that was developed, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (dec-RVKR-cmk, or CMK) was less cytotoxic and is cell permeable and has been used in many experimental studies [86, 106, 125]. It was recently shown to reduce the incidence of skin cancer in transgenic mice by inhibiting PACE4 as well as other PCs [126]. However a limitation of CMK’s use is that it is not furin specific, and is also known to inhibit other proprotein convertases [86, 102, 127]. Zhu et al. [127] has recently developed an antibody-based single domain nanobody which is a furin specific inhibitor. Through the use of this and other furin-specific inhibitors, it will be possible to delineate the role furin plays in the processing of specific substrates within in the cell. This will help in development of specific inhibitors, which will have therapeutic potential in the treatment of a variety of diseases.
Furin and other PCs have been shown to be involved in the maturation of both TACE and MMP within skin cells. ProTACE is processed by both furin and PC7 to its mature form thereby increasing its proteolytic activity [83, 85]. The maturation of TACE occurs as it transits through the Golgi compartment where the prodomain was removed by a furin-type proprotein convertase [77, 84, 85]. As increased amounts of mature TACE are detected in furin over-expressing cells, it appears that proTACE is a better substrate for furin than it is for PC7 [85]. A similar observation has been seen in cells overexpressing TACE [58, 83, 99] where furin was shown to be responsible for its cleavage [83, 99]. This finding was confirmed using cell permeable furin inhibitors CMK and PDX in Cos7 cells [83] and keratinocytes [98] where reduced levels of mature TACE were formed.
Furin mRNA, protein and enzyme activity has been observed in human epidermal keratinocytes [29, 98, 111, 128, 129]. Skiba et al. [29] found that UVA and UVB radiation immediately increased furin mRNA levels in HaCaT cells. UVB irradiation induced higher levels of furin mRNA expression [29]. The time course for furin mRNA levels in cells irradiated with low dose of UVA or high dose of UVB was similar to that for TNFα, whereas maximal mRNA induction of both genes were detected 8 h post-irradiation [29]. Although UV irradiation does appear to have an effect on furin gene expression, no direct relationship was apparent between TACE and furin mRNA induction. A recent study has shown that following exposure to UVA and UVB, furin levels in HaCaT cells fell with respect to time [49, 129]. However, it was unknown whether this was due to the loss of the pro or mature form of the enzyme. Through its effect on stimulating MMPs, as well as activating TACE and the resultant effect this has on TNFα released by the cell, furin activity has an influence on the inflammation seen in the skin following exposure to UV radiation as seen in Figure 2.
The role furin plays in the maturation of TACE and MMPs in skin cells. Furin cleaves and activates TACE, which in turn can process TNFα from its proform. Keratinocytes secrete TNFα following exposure to UVB radiation, and this is enhanced if IL-1α is present. Furin also cleaves MMPs from their respective proforms, and the expression and activity of these proteases are elevated when the cells have been exposed to UVB radiation, and they are enhanced if either IL-1α (MMP-9) or TNFα (MMP-2) is present. The effect of UVB radiation on the expression of the enzymes and pTNFα in the cell is represented by dashed lines, if it is enhanced it is represented by (+), and if it is unknown (?)
Furin/PC processing of substrates has been shown to also contribute to tumour progression, aggressiveness, metastasis, and angiogenesis [23, 24, 104-106]. Tumour invasion and metastasis represent a multistep process that depends on the activity of many proteins [46, 101, 104, 130]. Proteolytic degradation of the ECM components is a central event of this process. Several classes of proteases, including MMPs, serine proteases and cysteine proteases have been implicated in the tumour cell invasive process [104, 130, 131]. Of these, MMPs appear to be primarily responsible for much of the ECM degradation observed during invasive processes [111, 130, 132-134]. They can contribute to tumour growth not only by degradation of the ECM but by the release of sequestered growth factors or the generation of bioactive fragments VEGF, bFGF or TGFβ, the suppression of tumour cell apoptosis and the destruction of immune-modulating chemokine gradients [131, 132, 135]. Furin also cleaves a number of MMPs from their proform, and activating them as a result [23, 86, 102, 105].
MMPs belong to the family of zinc-dependent endopeptidases collectively referred to as metzincins. The metzincins can be subdivided into four families: seralysins, astacins, ADAMs/adamalysins, and MMPs [130, 136]. So far to date, 28 members of the MMP family have been identified [130, 135, 136] which are primarily responsible for most of the ECM degradation observed during the invasive processes. MMPs are produced by skin cells (fibroblasts, keratinocytes, melanocytes) as well as macrophages, endothelial cells and mast cells [10, 25, 81, 137]. MMPs are also implicated in cell migration, proliferation, and tissue remodelling and thereby may also play a role in growth and development, angiogenesis, and atherosclerosis [138, 139].
Structurally MMPs consist of a signal peptide followed by pro, catalytic, hemopexin and cytoplasmic domains, respectively [130]. MMPs cleave peptides and proteins, which have a myriad of functions that are independent of their proteolytic activity [140]. They have distinct but often overlapping substrate specificities, hence leading to the absence of distinct phenotypes in most genetically-engineered mice with knockdown of specific MMPs [140].
MMPs are generally expressed in very low amounts and their transcription is tightly regulated either positively or negatively by cytokines and growth factors such as IL-1, IL-4, IL-6, TGFβ, or TNFα [130, 135, 141, 142]. Some of these regulatory molecules can be proteolytically activated or inactivated by MMPs (via a feedback loop). MMPs are synthesized as latent proenzymes, which are converted into mature, catalytically active forms in the TGN by PCs [111, 139]. Activation of MMPs following secretion from the cell depends on disruption of the prodomain interaction with the catalytic site, which may occur either by conformational changes or proteolytic removal of the prodomain. With the exception of MMP-2, the mechanism for in vivo activation of secreted MMPs is not well understood [135].
In normal skin, MMPs are not constitutively expressed but can be induced temporarily in response to exogenous signals such as UVR [10, 25, 143]. Elevated levels of MMP activity in human skin, as a result of prolonged periods of sun exposure, confirm that it plays a major role in photoageing [10, 25, 28]. Onoue et al. [144] suggested that MMP-9 secreted from keratinocytes after UVB irradiation might result from apoptotic events. UV radiation is known to elevate the expression of MMP-1, MMP-3 (stromelysin-1) and MMP-9 in human skin in vivo [25]. All three MMPs (1, 3 and 9) can degrade most of the proteins found in the extracellular matrix [25]. MMP-1, which is produced by both dermal fibroblasts and epidermal keratinocytes, cleaves type 1 collagen into specific fragments. These fragments can then be further hydrolysed by MMP-2 and MMP-9 [137, 145]. Steinbrenner et al. [137] found that UVA irradiation dose-dependently decreased the steady-state mRNA levels of MMP-2 and MMP-9 and lowered the gelatinolytic activity of both enzymes in cell culture supernatants. Of interest is that in vivo, following exposure to UV radiation only keratinocytes express large levels of MMP on the cell membrane [146], but when fibroblasts grown in culture are irradiated they express higher levels of MMP on their plasma membrane [147]. The reasons for the discrepancies between the responses of human skin cells under in vivo and in vitro conditions are not known.
TNFα has been shown to induce proMMP-2 in human dermal fibroblasts [8], while IL-1α induced proMMP-9 levels in fibroblasts and keratinocytes [148]. In mesenchymal cells TNFα was shown to stimulate MMP-2 activity by activating a proteolytic cascade involving furin and MT1-MMP [139]. It is not known if TNFα activates MMP activity in epidermal cells via a similar mechanism. MMP-9 [144], has been shown to play an important role in the pathophysiologies of many skin conditions such as wound healing [145], and angiogenesis [87]. Activation of pro-MMP2 takes place at the cell surface and involves interactions with active MT1-MMP, which is itself activated through rapid trafficking to the cell surface and proteolytic processing [139]. Maquoi et al. [114] demonstrated that furin-inhibitor reduces the level of mature MT1-MMP, which is paralleled by a decrease in pro-MMP-2 activation as well as in cell invasiveness, confirming the furin plays a role in this process. Direct cleavage of proMMP-2 by furin in the TGN has shown to inactivate this matrix metalloprotease [149]. Therefore changes to the level of MMP-2 activity on the surface of the cell can be directly or indirectly regulated by furin either through cleavage in the TGN to reduce activity or indirectly via MT1-MMP, which increases activity. The mechanism by which this is regulated is not clear.
Low doses of UV radiation while they are not inflammatory, can cause mutations in skin tissue [9, 19, 30] or in cultured fibroblasts [150]. Constant exposure to UV radiation will not only result in mutations in p53 [150, 151] but in other genes including p16, PTCH, B-Raf, which will result in these cells becoming carcinogenic [6, 152, 153]. Apart from causing these mutations, UV radiation enhances the release of inflammatory molecules (e.g. TNFα, IL-1α, ROS, chemokines) from the skin (see section 2 for details). The result of which is the creation of an environment that allows for these mutated skin cells to become cancerous over time [15, 19, 21, 46, 56]. As a result of increased levels of MMP activity on the cell membrane, either as a result of UV exposure or other factors, these skin cancer cells may become metastatic as a result of epithelial to mesenchymal transition (EMT) [154].
In the progression of a mutated cell to that of a tumorigenic or metastatic cell, PCs have been shown to cleave a range of precursors of growth factors, their receptors, adhesion molecules, proteases and MMPs. Some of these molecules include cadherins, TGFβ, platelet derived growth factor as well as insulin-like growth factor 1 and its receptor [23, 101, 106, 117, 155]. Tumour progression and metastasis may enhanced by a number of factors such as (a) hypoxia-induced upregulation of furin activity within the solid tumour mass [105], (b) changes in cell adhesion through PC cleavage and activation of integrins and related adhesion molecules [156], (c) furin processing of vascular endothelial growth factor resulting in increased angiogenic activity [157], and (d) furin enhanced expression of MMPs [84, 158].
Furin/PC expression and processing can increase the incidence and severity of the cancer phenotype [104, 111]. Aberrant furin expression has been observed in a number of tumours including those from the breast [159], ovary [160], liver [125], brain [161], skin [111, 132] and from other tissues [23, 128, 162, 163]. Bassi et al. [101] observed that PACE4 transgenic mice were more susceptible to epidermal carcinogenesis and tumour progression compared to controls. These transgenic keratinocytes had higher rates of processing of MT1-MMP and MT2-MMP resulting in increased collagen degradation.
MMP-2 (gelatinase A) and MMP-9 (gelatinase B) have been frequently associated with the invasive and metastatic potential of tumour cells [10, 104, 111, 130, 132, 133, 137]. The expression of MMP-2, is regulated independently of MMP-9 [144]. The close correlation observed between MMP-2 activation and metastatic progression in various tumours suggests that it may act as a “master switch” triggering tumour spread [114]. The expression of MMP is low in keratinocytes but elevated in BCC and SCC [49, 164, 165]. In SCC, MMP proteins (1, 2, 3 and MT1-MMP) are expressed both in tumorous and stromal cells [165], while MMP proteins (1, 2, 3 and 9) are observed in BCCs and melanomas [165, 166]. This expression correlates with the progression and the metastatic potentials of these tumours [106, 135, 165, 167]. UVR can participate to the development of skin cancer by the activation of MMPs. Two molecular mechanisms contribute to the UV-induced MMPs expression. First, the activation of cell-surface receptors with subsequent activation of mitogen activated protein kinase (MAPK) cascade that in turn contributes to the transcriptional up-regulation of MMPs [168]. Second, through the expression of pro-inflammatory cytokines, which induce the expression of MMPs [46, 133, 142, 143]. The role of UV in the induction of MMPs is supported by two experimental findings. First, UV-irradiation of SCC cell lines results in an increased secretion of MMPs [49, 132]. Second, UV-induced phosphorylation of extracellular signal-regulated kinase (ERK) and stress kinases precedes the rapid stimulation of MMPs in SCC cells [10]. If there are cells in the skin which become cancerous as a result of DNA damage, some may go onto become metastatic due to increased MMP activity [111]. This increase in levels of activated MMPs on the surface of the cell could be due to either increased expression of proMMP protein and/or increased furin activity. The role furin plays in the development of skin cancer suggests that it could be significant, and as such the development of specific inhibitors may offer a new therapy to treat such tumours.
Recently, Fu et al. [106] showed that in transgenic mice overexpressing furin, exposed to chemical carcinogens formed more and larger tumours than did control mice. This suggests that furin enhances skin cancer growth. Of interest was that these tumours were induced by chemical carcinogens and not by UV radiation. While enhanced furin levels were shown to enhance skin tumour development, in studies using cultured melanoma and glioma cells, furin inhibition was shown to reduce the processing of the pro-N-cadherin adhesion molecule, enhancing their migratory and aggressive nature [118]. The result of this study suggests that high furin levels may not enhance the metastasis of some tumours. Huang et al. [125] when investigating the expression of furin in hepatocellular carcinoma and surrounding tissue in humans, observed that if this ratio was >3.5 this resulted in higher patient survival rates compared to those who had ratios <3.5. This suggests that furin may play a dual role in cancer development, though the exact nature of which is not clear at this stage. Further study on this is warranted, and may result in a better understanding of its role in cancer development, and from which it may be possible to develop specific furin inhibitors which could be used clinically to treat these tumours.
While it has been shown that UVA and UVB radiation cause different effects on the immune response this could be related to the activity of cell surface metalloproteases found on skin cells. Although the effect of TNFα in UV-induced inflammation has been well documented, little is known if the changes in TACE activity are due to increased protein levels or changes in enzyme activity. The inflammatory environment, seen in the skin, following exposure to UV radiation is known to stimulate the development of mutated cells which possess DNA damage caused directly by UV radiation or indirectly through the generation of ROS. Apart from processing TNFα, TACE also cleaves EGF family members, which would stimulate the growth of these mutated cells, which over time may become cancerous. As TNFα is a powerful inflammatory cytokine, considerable research has been under taken to develop specific TACE inhibitors [55, 56, 80]. Such inhibitors may play an important role in preventing the development of UV-induced skin cancers. An increased knowledge of the roles played by metalloproteases in tumour progression, combined with the use of more selective inhibitors, could lead to effective use of these compounds in cancer therapies [55].
Similar to that of TACE, MMPs are also activated by UV radiation and also play a crucial role in skin tumour cell development and metastasis. Furin and other PCs have been shown to play an important role in activating both TACE and many MMPs in skin cells. Whereas the overexpression and activity of furin exacerbates the cancer phenotype, inhibition of its activity decreases or nullifies its effects, and thus, the development and use of specific inhibitors may also be a viable route to cancer therapy [23, 86, 102, 127].
It is known that UV induces furin mRNA in skin cells, though protein levels do not appear to change with respect to time, which suggests a rapid turnover of the enzyme. Further study is needed on how UV radiation activates furin, TACE and MMPs in skin cells. These proteases play an important role in the changes observed in those epidermal cells exposed to UV radiation. The development of specific furin inhibitors has the potential to reduce the carcinogenic effects of sunlight by preventing the activation of TACE and MMPs and their subsequent downstream effects. Such compounds may have the potential to offer new therapies both in the prevention and treatment of skin cancer.
Monitoring of water pollution is very important for the preservation of the environment and prevention of negative impacts that it can have on human health. Therefore, great attention is paid to simplifying procedures for detection and monitoring of pollutants. Heavy metals are particularly dangerous due to their ability to accumulate over time in both plants and animals, as well as in water. For these reasons, there are already developed different methods that determine their concentrations generally in the environment.
Biosensors represent a simple, reliable, and fast solution for monitoring water pollution caused by various heavy metals. The small size of biosensor devices has enabled their in situ application, thus avoiding long-term and sometimes expensive measurements in laboratories.
According to IUPAC, biosensor represents a “self-contained integrated device, which is capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element (biochemical receptor) which is retained in a direct spatial contact with an electrochemical transduction element” [1, 2]. Biosensors allow not only determining the presence and overall biologically available concentrations of heavy metals in water but also assessing their biological effects, such as toxicity or cytotoxicity, which are sometimes more important than chemical composition information.
The term “heavy metals” refers to all metals except Al, Na, Ca, Mg, and K, i.e., to all metals that have a density higher than 5 g/cm3. It includes a number of physiologically important elements such as Fe, Cu, Zn and Mn, then highly toxic Pb, As, Hg, Cd, Sb, Cr(VI) and less toxic Au, Ag, Mo, Cr(III) and Co [3]. The physiological and toxicological effects of these elements represent a collection of very different mechanisms.
Even at very low concentrations, they pose a threat to the environment and human health, because they are not biodegradable, so heavy metals are the cause of one of the most serious pollution problems. The most important nonessential heavy metals which affect the surface water systems are cadmium, chromium, mercury, lead, arsenic, and antimony [4].
Heavy metals present in pesticides and therapeutic agents are additional pollution sources. Burning of fossil fuels containing heavy metals and increasing industrial applications of metals such as metal galvanizing, paint and varnish industry, and mining and chemical industries are the main source of pollution of water systems by heavy metals.
Heavy metals are transported with waste water at the place of discharge and contaminate water sources downstream from an industrial site. In water, heavy metals have the ability to bind to the surface of microorganisms, from where they are transported inside the cell where they can be involved in chemical reactions and change chemically.
The majority of known techniques can determine the total amount of heavy metal ions. In addition, laboratory techniques that are routinely used for the analysis of metal ions, such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, anodic stripping voltammetry, and X-ray fluorescence spectrometry, require sophisticated equipment, pretreatment of samples, or qualified operators.
However, today it is known that only certain oxidation states of biologically available metal ions pose the greatest risk to human health and the environment. For example, “Cr(III) is an essential nutrient required in insulin action and sugar and fat metabolism, while Cr(VI) is believed to be highly toxic and carcinogenic” [5].
Metals and metalloid ions can be divided into three groups according to their toxicity. The first group includes metals (metalloids) that are toxic at extremely low concentration, such as lead, cadmium, and mercury. “Metals of the second group (arsenic, bismuth, indium, antimony and thallium) are less toxic, i.e., they are toxic only in higher concentrations. The third group includes metals (metalloids) of essential importance, such as copper, zinc, cobalt, selenium and iron, which are necessary for different chemical and biochemical processes in the body, and are toxic only above a certain concentration.” Concentration window “of these heavy metals is somewhere between toxic and maximum permissible limits” [6].
Table 1 gives critical concentrations of some heavy metals in natural waters according to EPA [7].
Metal | Max. allowable concentration (μg/ml) |
---|---|
Mercury | 0.002 |
Arsenic | 0.5 |
Lead | 0.5 |
Copper | 0.6 |
Cadmium | 0.04 |
Zinc | 5 |
Critical concentrations of some heavy metals in natural waters according to EPA
The toxic effects of heavy metals can be the result of changes in numerous physiological processes at the cellular or molecular level caused by the inactivation of the enzyme. It can also occur as a result of the blocking of functional groups of metabolically important molecules or by replacing the essential elements and disturbing the integrity of the membrane. A rather frequent consequence of heavy metal poisoning is the production of reactive oxygen species (ROS) due to interference with the transport activities of electrons, especially the chloroplast membrane [8]. This increase in ROS exposes cells to oxidative stress that leads to peroxidation of lipids, biological damage of macromolecules, membrane decay, and DNA splitting [9].
They can penetrate into the organism in elemental form, in salt form, or as organometallic compounds, wherein the process of absorption, distribution, deposition, and elimination depends on the form in which the metal is present. Metals are very toxic because they are either in ionic form or within the compound, soluble in water, and easily absorbed by living organisms [3].
The mobility of heavy metals in water is particularly affected by the pH of water, the presence of hydrated forms of Mn and Fe, the concentration of carbonates and phosphates, as well as the content of organic matter. In addition, if the medium is very acidic and increased redox potential, the mobilization of Cu and Pb occurs, and under the reduction conditions, the hydroxides Mn and Fe are mobilized.
Heavy metals which are mostly the subject of research and monitoring in water and also generally in the environment due to their pronounced toxicity are arsenic, chromium, lead, mercury, and cadmium, while zinc, cobalt, copper, iron, and manganese are also interesting because they belong to the group of essential elements. The level of toxicity for some of these heavy metals is at or slightly above the concentration in which they are naturally found in nature [10]. Heavy metals occur in the environment naturally or as a result of human activities. Natural sources include volcanic eruptions, weathering (acid rock drainage), and discharge into rivers, lakes, and oceans.
Anthropogenic sources of heavy metals have emerged with the development of society. For example, the release of metal from the dishes causes contamination of food and water with metals.
Iron belongs to a group of essential metals and is crucial for a number of synthetic and enzyme processes in the human body. Most of the iron in our body exists as part of the hemoglobin molecule or myoglobin molecule. In addition to the vital importance it has for most living organisms, iron is potentially toxic at high concentrations. The effect of iron on aquatic organisms and their habitats is mostly indirect. Combined direct and indirect effects of contamination of the aquatic environment cause a decrease in biodiversity and number of fish. In aqueous solutions, the Fe3+ ion is in the form of the aqua complex, Fe(H2O)63+, which is quite hydrolyzed (hydrolysis starts at pH 1). Hydrolysis of Fe(III) ions depends on the type of ionic environment, temperature, and the presence of other substances. The results of the researches show that the most important chemical types are found in hydrolyzed solution.
Copper is a microelement of outstanding biological importance and is part of essential metabolic pathways. Copper ions play a key role in active centers of oxidoreductases, such as superoxide dismutase (Cu, Zn-SOD), [5], an enzyme important for maintaining a low level of free radicals in the cell, thus protecting biomolecules such as proteins and lipids from the pathological conditions.
Copper deficiency can cause anemia, because insufficient amount of copper causes poor absorption of iron, reducing the number of red blood cells. The lack of copper also reduces the amount of white blood cells and therefore the resistance of the organism to diseases. In general, copper is not considered to be a major ecotoxicological problem, but its widespread distribution and exposure to exhaust gases are certainly the reasons why copper is involved in the structuring of ecosystems. Copper is found in three oxidation states, Cu+, Cu2+, and Cu3+, with the Cu2+ form being the most common. The most mobile forms of copper are Cu2+ and CuOH+. In the aqueous environment, copper is found in three basic forms, as suspended, colloidal, and dissolved. The accumulation of copper in the aquatic environment results in the primary exposure of aquatic organisms. Aquatic organisms can accumulate dissolved copper by direct absorption through the body surface, while colloidal forms of this metal are introduced into the body by ingesting contaminated food.
Zinc participates in the structure of many enzymes and is an essential element. It is attached to insulin and plays a significant role in the metabolism of nucleic acids and amino acids, DNA replication, and gene expression. However, like all other essential metals, zinc in higher concentrations is toxic to living organisms. Zinc can bioaccumulate in fish, and the degree of bioaccumulation usually depends on the exposure mode, as well as the conditions prevailing in the observed aquatic environment. Conditions that may affect the toxicity of zinc (but also other heavy metals) in the aquatic environment are the content of Ca and Mg, the pH of water, the content of the hydroxide (alkalinity), and the content of dissolved natural organic matter, i.e., humic substances.
The required amount of cobalt in the body is about 5 mg for vitamin B12 to avoid anemia. In general, cobalt has low toxicity. Gastrointestinal (digestive tract) absorption of soluble cobalt compounds is estimated to be 25%. However, cobalt is toxic to humans. When cobalt has been used as an additive in beer (for foam stabilization), severe biventricular heart failure and a high mortality rate were observed in heavy beer drinkers [11].
Long-term inhalation of cobalt dust irritates the respiratory tract and can cause chronic bronchitis, and cobalt salts can cause benign dermatosis. Cobalt occurs in oxidation states 0, +1, +2, +3, and +4, and most of its compounds have an oxidation number +2 and +3, of which the cobalt(II) compounds are more stable. Most cobalt(II) compounds have an ionic character (halides and numerous Co(III) complexes). Cobalt is relatively a nonreactive metal. It does not oxidize under dry and humid conditions at normal temperatures. It binds to halogen elements by heating. Cobalt is used in the production of artificial fertilizers and so can be found in higher concentrations in soil and water. It is also used in medicine, in the treatment of anemia that cannot be treated with iron.
Lead in the environment mainly comes from anthropogenic sources such as combustion of fossil fuels, landfills and fires at landfills, waste industrial sludges, phosphate-based fertilizers, pesticides, and exhaust gases from vehicles.
It is found in the form of sulphates, sulphides, and carbonates. It is considered the leading environmental pollutant and is increasingly endangering the living world, especially the surrounding areas of large industrial plants, frequent roads, and large cities.
The intensity of the adoption of lead depends on its concentrations in soil, soil pH, organic matter content, ratio of cations and anions, and other environmental factors. Human is exposed to toxic effects of lead by consuming food and water that are contaminated with this heavy metal but also by inhaling particulate matter with lead content. Absorption over the skin is only possible for tetraethyl and tetramethyl lead. Lead is rapidly absorbed into the bloodstream and binds to red blood cells in the form of Pb2+, and via blood about 90% is deposited in the bones in the form of Pb3(PO4)2. In the case of acidosis (increased acidity), the mobility of lead from the bones in the form of Pb2+ which has a toxic effect on the central nervous, circulatory, and immunological systems and kidneys can occur. [10]
Mercury vapors and organic compounds of mercury are very strong poisons. Harmful substances are released by combustion of fossil fuels, and the risk of pollution threatens also due to increased use of mercury in industry and agriculture [12].
In its compounds, chromium exists in several oxidation states: from bivalent to hexavalent. In solutions, chromium can occur in trivalent and hexavalent forms. Hexavalent chromium is usually present in the compounds as chromate (CrO4)2− or dichromate (Cr2O7)2− ion. Cr(VI) is toxic due to its high degree of oxidation and easily enters the biological membranes. Therefore, this form of chromium is considered carcinogenic. Because chromium(VI) is toxic, carcinogenic, and mutagenic to living organisms, damages the liver, and causes lung congestion, skin irritation, and the formation of ulcer, it needs to be removed from the wastewater before their release into natural recipients. On the other hand, trivalent chromium, Cr(III), is 300 times less toxic than chromium(VI). Chromium is a vital nutrient for many animal and plant species, but it can also cause allergic reactions on the skin and can be carcinogenic [13].
A biosensor is an analytical device consisting of immobilized biological material in direct contact with a compatible transducer that will convert the biochemical signal into a measurable electrical signal. Biomolecules are responsible for specific recognition of the analyte, while the physicochemical converter provides electrical output signal that is amplified by electronic component [14]. Biosensors find application in various areas, from agriculture, food quality control, medicine, army, and control of various processes in the environment. Biosensors can provide quick information about the site of pollution, which is necessary for environmental control and monitoring. In addition, the advantage of biosensors over other analytical methods is their mobility that allows researchers to measure the in situ pollutant concentration and the ability to measure the concentration of pollutants in situ without additional sample preparation. Also, in addition to the determination of specific compounds, they can provide information on their biological effect (e.g., toxicity of a compound).
Due to exceptional performances, including high specificity and sensitivity, rapid response, low cost, relatively small size, and simple operation, biosensors have become an important tool for detecting chemical and biological components and their monitoring for clinical, nutritional, and ecological needs [15].
Biosensors are analytical sensory devices that combine physical and chemical sensing techniques [16, 17]. Their performance is based on direct contact of two elements: biological and physicochemical, whose tight bond is achieved by physical or chemical methods of immobilization. Biological element serves as a receptor (bioreceptor), i.e., for the recognition of particular analyte from the medium of interest, based on the interaction of analyte and bioreceptor. Physicochemical transducer converts the response that occurs as a result of analyte-bioreceptor interaction on their interface into a measurable signal which can be processed and displayed in the form of readable values. For proper biosensor operation, the biological compound has to be immobilized in the vicinity of the transducer, and immobilization can be done either by physical entrapment or chemical attachment. Only small amounts of bioreceptor molecules are required, and they will be repeatedly used for measurements [18].
The displayed values are in correlation with the detected analyte-bioreceptor interactions, i.e., the concentration of a specific analyte or group of analytes in the analyzed sample [4, 16, 17]. General working principle of biosensors is illustrated in Figure 1.
Schematic illustration of a biosensor general working principle.
Although widely used, conventional analytical techniques require sophisticated instruments and highly trained personnel to conduct operational procedures and sample preparation, which makes them expensive and time-consuming [19, 20], thus not enabling determination of a large number of samples in a short time [21].
The main advantages of biosensors in relation to conventional analytical techniques are possibility of miniaturization and portability of device, reduced requirements for laboratory skills, reduced sample volume and pretreatment [1, 22], assessment of all possible types of analytes, inorganic or organic [23, 24], and possibility of performing single measurements or continuous real-time monitoring of analytes [1, 25]. Biosensors allow estimation of biological effects, e.g., toxicity of specific chemicals, because they can be used to detect their bioavailable concentrations [26].
Biosensors can be divided into classes according to different approaches, among which the two are commonly used—type of biorecognition element (biocomponent, bioreceptor) and type of transduction system in biosensor. Each class of biosensors can be further classified into subclasses (Figure 2).
Schematic illustration of the common classification of biosensors.
Based on the principle used in transduction systems, electrochemical, optical, piezoelectric, and thermal biosensors may be distinguished.
The first proposed and commercialized biosensors were electrochemical biosensors, which is why they are most commonly reported. The basic principle of this class of biosensors is that the interaction between the biomolecule (bioreceptor) and the target analyte results in a chemical reaction that produces or consumes ions or electrons and in turn changes the electrical properties of the analyte solution, such as electrical current or potential. Transducer detects these changes by producing an electrochemical signal which is correlated with the amount of analyte present in the sample solution.
Advantages of electrochemical biosensors include minimal requirements for sample preparation and sensitivity at small sample volumes. It is also possible to perform sample analysis directly, which enables automation. Drawbacks of detections are poor reproducibility and stability [27].
Electrochemical biosensors are classified according to the type of measured signal into subclasses: potentiometric, amperometric, conductometric, and biosensors based on ion-selective field-effect transistors (ISFETs). Different measurement principles always require a specific design of an electrochemical cell [21].
Potentiometric biosensors are based on the use of ion-selective electrode (ISEs) at the top of which an ion-selective membrane is placed which is responsible for selectivity to target ions in the presence of interfering ions in the sample. These devices measure the difference between the potential of the working and reference electrodes at essentially zero current, and this difference corresponds to the concentration of the analyte.
Amperometric biosensors are the most widespread class of electrochemical biosensors. Amperometric biosensors are more sensitive and faster than potentiometric but have poor selectivity and are susceptible to the interference of electroactive species that are not of interest [22, 28].
Conductometric biosensors are based on measurement of electrical conductivity in sample solution between two electrodes, as a consequence of the biochemical reaction. Conductometric biosensors operate at sufficiently low driving voltage, are not sensitive to light, do not require the use of a reference electrode, and can be produced using inexpensive technology [23, 24].
Biosensors based on the ion-selective field-effect transistors (ISFET) are the fourth class of electrochemical biosensors, suitable for the direct detection of ions. Change of activity of ions of a sample causes a change in the potential of the gate electrode that is brought into contact with the analyte solution. The change of the electric potential is then measured.
Optical biosensors are a biosensor class in which the transducer detects optical changes in the input light resulting from the interaction of the bioreceptor and the target analyte, and the amplitude of these changes is in correlation with the concentration of the present analyte in the analyzed sample. Among the significant advantages of these optical devices are insensitivity to electromagnetic interference, small instrumentation, simplicity, and noninvasiveness of measurement, as well as the possibility of application in vivo, since they are non-electrical biosensors. According to the optical configuration, biosensors can be intrinsic or extrinsic. In intrinsic biosensors, the incident light wave is closed in a wave guide or an optical fiber, along which it propagates, but the design of the structure in which the wave is closed is such that it allows the interaction of the wave with the analyte. In extrinsic biosensors, the light wave passes directly through the sample phase and reacts with it, and the optical fiber serves as a means of transmitting the signal.
Absorption-based biosensors are simple and inexpensive devices that allow the determination of concentrations of different analytes, based on the fact that each type of analyte absorbs a certain wavelength of light emitted into the sample. Guiding the light from the light source to the sample and from the sample to the detector can be performed using the same optical fiber or different fibers [29].
Surface plasmon resonance (SPR) biosensors use an optical detection technique where on the interface of metal and dielectrics, the amplified incident light hits the metal surface and excites the electrons, thereby generating electromagnetic waves (plasmons). Plasmon propagation is very sensitive to the changes in the refractive index of the material near the metal surface, which are caused by biomolecular interaction, such as, for example, specific binding of the analyte [30, 31].
Fluorescence-based optical biosensors can directly detect target atoms or molecules by measuring the change in the frequency of electromagnetic radiation emitted by them. The frequency change is stimulated by the absorption of radiation and the consequent appearance of the excited state of the target species. Detection can also be carried out indirectly, using fluorescence labels or fluorescence energy transfer (FRET).
Luminescence-based biosensors can be classified into chemiluminescent and bioluminescent optical biosensors. Unlike biosensors based on fluorescence, in these sensor devices, the triggered state of the target atoms or molecules is obtained as a result of their exothermic chemical reaction, and while returning to the ground state, the excited species emit light without or with minimal heat. When such a chemical reaction occurs within a biological organism, then it is a bioluminescence.
Piezoelectric biosensors are devices in which the biorecognition element is integrated with a piezoelectric material used as a transducer. Among many types of natural and synthetic materials that exhibit a piezoelectric effect, quartz crystals are most commonly used [28, 32] because of their availability, as well as high temperature resistance and chemical stability in aqueous solution. The basic principle of measurement for this type of biosensor is based on the ability of a piezoelectric material to generate electrical potential when deformed under the applied mechanical stress, and vice versa, to elastically deform when exposed to an electric field.
Thermal biosensors, also called calorimetric or thermometric, are a biosensor class in which the transducer detects interactions between bioreceptors and analyte resulting in a change of temperature, which is in correlation with the concentration of the analyte. As thermal transducers in these devices, thermistors or thermopiles are used [21, 33]. Some of the advantages of thermal biosensors are detection without the need for labeling of reactants, not requiring frequent recalibration, and no disturbances by electrochemical and optical properties of the sample [21, 34]. In most research papers published about this type of sensor, described experiments were carried out using enzyme-based thermal biosensors, due to the exothermic nature of the reactions catalyzed by enzymes.
Biocomponent/bioreceptor is responsible for the detection and interaction with the analyte and therefore is a very important part of any type of biosensor. The receptor is responsible for the selective and sensitive recognitions of the analyte, and the energy liberated during the interaction of the analyte and the receptor is converted into an electrical signal that is suitable for measurement. The most commonly used biological elements are enzymes and antibodies. Biosensors can be divided into two main categories: biocatalytic and affinity sensors based on the interaction between biological material and analyte.
Biocatalytic biosensors, also known as metabolism sensors, comprise a biological component that catalyzes the chemical conversion of the analyte with which it interacts and detect the magnitude of the resulting changes such as product formation, reactant disappearance, or inhibition of the reaction, which are correlated with the concentration of the analyte [35]. Affinity biosensors are based on selective interaction between the analyte and the biological component through their irreversible binding, resulting in a physicochemical change detected by the converter.
Antibodies are proteins produced by the immune system in response to a foreign substance in the body. Also known as immunoglobulins (Ig), they are Y-shaped proteins generated by a type of white blood cells called B lymphocytes (B cells). Their ability to recognize specific molecules makes them suitable for use as biorecognition component in biosensors. During the process of biological recognition, the antibodies bind tightly to antigens forming complexes. There are five classes of antibodies, based on their structure and function: IgA, IgD, IgE, IgG, and IgM. Among them, IgG is the class most frequently used for heavy metal detection, because of their higher affinity and specificity compared to other classes. Antibodies such as monoclonal, polyclonal, or recombinant can be utilized in biosensors. Monoclonal antibodies are homogeneous antibodies, derived from single B cell; thus they all have the same specificity, i.e., to bind to one unique epitope (binding site) on a specific antigen. Unlike monoclonal antibodies, polyclonal antibodies are produced from different B cells against the same antigen and therefore have affinity for various binding sites of that antigen. This feature of polyclonal antibodies results in their stronger binding to the target species, but due to the recognition of multiple epitopes, they have higher potential for cross-reactivity, i.e., specificity for nontargeted antigens with similar structural regions as the targeted one. The production of recombinant antibodies is enabled by genetic engineering. Important properties of antibodies for providing accurate results for detection and measurement using biosensors are high sensitivity and specificity, with minimal cross-reactivity [36].
Different types of approaches have been developed and used for immobilization of Abs onto a sensor surface, such as covalent binding, non-covalent immobilization, and coupling by affinity interactions, because the immobilization is the crucial step which can affect the optimal performance of an antibody-based biosensor [37]. Reaction conditions, such as temperature, pH, and ionic strength, can also affect the activity of the antibodies [38].
Enzymes are biocatalysts that catalyze chemical reactions. Their task is to translate the characteristic substance (substrate) into a product. Enzymes are highly selective for the particular substrate which makes them suitable sensor material. Detection mechanism of enzyme-based biosensors is based on activation or inhibition of their activities as a response caused by heavy metals. Usually the metal ion reacts with the thiol groups present in enzymatic structures that result in conformational changes and thus affect the catalytic activity. Different enzymes have been used for the structure of biosensors based on inhibition. Enzymes such as glucose oxidase, urease, glutathione S-transferase, alkaline phosphatase, lactate dehydrogenase, acid phosphatase, and invertase have been utilized to detect metals such as cadmium, lead, copper, mercury, zinc, etc. However, inhibition-based biosensors have an important disadvantage, which is insufficient selectivity because some of the enzymes simultaneously inhibit several metals.
Biosensors based on immobilized enzymes are also used, and they show several advantages compared to free enzymes:
A thousand times lower consumption of immobilized enzymes.
Reduced interferences in differential mode.
No preincubation is required.
Faster analysis, less than 5 min.
In the case of reversible inhibition, sometimes reactivation of the enzyme activity is not necessary.
The problem with biosensors based on enzymatic inhibition is that only a few enzymes are sensitive to heavy metals.
Proteins, such as phytochelatins or metallothioneins, can be used as biological components in biosensors when immobilized on the surface of the transducer [39]. The interaction of proteins and metals in the biosensor is realized through the formation of complexes, and the detection technique does not require labeling. The resulting changes in the protein layer are detected by measuring the electrical capacity or impedance by the relevant transducer. Using the protein biosensor enabled the assessment of bioavailable concentrations of heavy metals. In addition, using capacitive sensors, which belong to the class of electrochemical biosensors, it is possible to achieve much higher sensitivity to low concentrations of heavy metals, compared to cell-based devices.
Whole cell-based biosensors are based on using biosensing cells, such as microorganisms, plant cells, algae, fungi, protozoa, etc., which can be natural or recombinant [40]. The use of whole cells as biological elements of recognition has many advantages. Whole cell-based biosensors are usually cheaper than biosensors based on enzymes, because the whole cells can be easily cultivated and are easier to isolate and purify compared with enzymes. Whole cells are more tolerant to a significant change in pH, temperature, or ionic strength. A multistep reaction is possible because one cell can contain all the enzymes and cofactors needed to detect the analyte. Biosensors of this type can easily be regenerated or maintained by allowing cells to regrow while working in situ. Preparation of samples is usually not necessary. Compared to enzyme-based biosensors, the disadvantages of these devices are that they are susceptible to interference of contaminants that are not targeted analytes. They also have a relatively slow response, compared to other types of biosensors.
The unique biosensor features make them widely applicable in the field of water quality control, from the point of view of detecting and determining the concentration of heavy metals. The use of biosensors for individual or continuous measurements is dependent on the type of biologically active element. Since biological compounds such as cholesterol, glucose, urea, etc. are generally not electroactive, the combination of reactions is needed for obtaining an electroactive element, which leads to a change of current intensity [41]. Table 2 shows the classification of biosensors based on the recognition component that was utilized for the detection of heavy metals.
Type of bioreceptor | Analyzed heavy metal | Reference |
---|---|---|
2A81G5 Antibody ISB4 12F6 | Cd Cd U | [42] [43] [44] |
Alkaline Phosphatase Pyruvate enzymes Oxidase Urease | Zn Hg Cd Hg Hg, Ag | [45] [46] [47] [48] |
Glutathione S-transferase Mer R proteins Metallothionein | Cd, Zn Hg, Cu, Cd, Zn, Pb Cd, Zn, Ni | [49, 50] [51, 52] [53] |
Whole cells and cardiac cells | Hg, Pb, Cd, Fe, Cu, Zn | [54] |
Classification of biosensors based on the recognition component that was utilized for the detection of heavy metals
A proper immobilization of the biosensing element onto the transducer surface maintains biomaterial functionality while ensuring accessibility of the receptor cells toward analytes and proximity of the bioreceptor and transducer. The factors which determine the choice of a suitable physical or chemical immobilization method are physicochemical properties of the analyte, nature of the chosen biosensing element, the type of used transducer, and the operating conditions of biosensor. Antibody-based biosensors can be used as an alternative approach for the detection of metal ions, due to antibody features such as high specificity and binding affinity for antigens harmful for the organism. Detection mechanism of these devices is based on antibody-metal ion complex formation. The resulted response of their immunochemical interaction is converted by a transducer to measurable values and processed to readable values. Antibodies are capable for antigen detection in very low concentrations [38], but if their cross-reactivity is high, they can yield false-positive results of an assay of heavy metals in water [55].
A monoclonal antibody that recognizes 16 different metal-EDTA complexes has been produced and evaluated in terms of its binding affinity. The obtained results showed that the antibody has a maximum binding affinity for cadmium and mercury-EDTA complexes. [56]. In the inhibition immunoassay where the measurement of Cd2+ in water samples was carried out using monoclonal antibodies firmly bound to the cadmium-EDTA complex, but not to EDTA without metal [42], the biosensor showed satisfactory insensitivity to cations Ca2+, Na2+, and K1+ it encountered and achieved a reliable measurement in the presence of 1 mM of excess Fe3+, Mg2+, and Pb2+.
Monoclonal antibodies were used to detect Pb2+ without labeling, in a localized surface plasmon resonance-based optical biosensor [57]. The results of the experiment showed that at optimal monoclonal antibody immobilizing conditions, absorbability increased to 12.2% for detecting 10–100 ppb Pb(II)-EDTA complex with a limit of detection of 0.27 ppb.
Kulkarni et al. were the first to develop acid phosphatase-based fluorescence biosensor for the analysis of heavy metal ions Hg2+, Cr2+, and Cu2+. Increased concentration of metal ions resulted in increased enzyme inhibition and therefore decreased fluorescence. The enzyme was stable for more than 2 months at 4°C [58]. They also observed that mixture of heavy metal ions exhibit positive effect on the performance of biosensor.
The urease enzyme has been widely investigated as a possible biocomponent in heavy metal detection biosensors. Urease has been tested single and in combination with other enzymes. Electrochemical biosensor based on urease and glutamic dehydrogenase (GLDH) was developed for detecting heavy metals in water samples [59]. Also, a disposable potentiometric biosensor based on pure urease was developed, with the ability to detect copper and silver at sub-ppm level. For the detection of Pb and Cd in liquid samples, biosensors based on the combination of urease and acetylcholinesterase (Ache) were developed as a biocomponent with a detection limit of 1 ppb in water samples. It is known that ions of heavy metals inhibit alkaline phosphatase which was used for forming the biosensor with alkaline phosphatase as a biocomponent. It was found that the sensitivity of the developed biosensor to Cd2+ and Zn2+ was 10 ppb, whereas, with regard to ion Pb2+, there was no significant inhibition.
Two protein-based biosensors were developed on the basis of GST-SmtA and MerR [60] proteins, and their sensitivity and selectivity for heavy metal ions (Cd2+, Cu2+, Hg2+, and Zn2+) were measured using a capacitance transducer. Both types of biosensors have shown high sensitivity, enabling detection of metal ions up to femtomolar concentration.
Capacitance protein-based biosensor using synthetic phytochelatins (ECs) was developed for the detection of heavy metal ions (Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+), and the results of the experiments showed a lower sensitivity for all metal ions except for Zn2+ compared to systems based on SmtA and MerR, which can be explained by conformational changes in the protein, taking into account that the change in capacitance is function of the resulting change in protein conformation [51].
In cell-based biosensors, bioelement is fused with reporter gene. The detection mechanism is based on the activation of the reporter gene upon the contact between bioreceptor and target analyte, yielding an output measurable signal that is a correlation with bioavailable concentration of heavy metal.
Various cell-based biosensors have been used for the detection of heavy metals in water due to their ease of production and field testing, the ability to perform fast single measurement, as well as continuous measurements, and the ease of identifying bioavailable concentrations of toxicants that allows estimation of effects that heavy metals have on living organisms.
The advantage of bacterial cells is resistance to environmental conditions that could destroy the sensory element if exposed to them, supplying it with a relatively stable environment. Due to specific metabolic pathways used in microorganisms, compared to isolated enzymes, microbial sensors have the potential for more selective analysis of heavy metals which cannot be measured by simple enzyme reactions [61].
In order to be available for any sensing mechanism that is based inside the cell, there is a need for analytes to be able to enter the cell via diffusion, nonspecific uptake, or active transport. Alternative approaches are implemented in the cases when membrane permeability for an analyte is not sufficient. These approaches include allocation of the recognition element to the outside of the cell or the introduction of an appropriate transport mechanism for importing the analyte [61].
A large number of studies in which performances of whole cell-based biosensors were tested have utilized electrochemical and optical transducers. For detection of heavy metal ions (Cd2+, Cu2+, Fe3+, Hg2+, Pb2+,and Zn2+) at concentrations of 10μM, a mammalian heart cells-based biosensor was developed [54], with excellent performance in terms of frequency selection, amplitude and duration of detection within 15 min.
Biosensor, based on immobilized engineered bacteria Alcaligenes eutrophus (AE1239) and optical transducer, was utilized for monitoring the bioavailable copper ions in synthetic water samples, wherein the lowest limit of detection was 1 μM [62].
Biosensors have a very wide range of applications, from environmental monitoring, food safety, detection of various diseases, use in artificial implantable devices such as pacemakers to the detection of drugs.
Application for pollution monitoring requires the biosensor to work from several hours to several days. Such biosensors are a tool for “long-term monitoring.” Whether it is a long-term follow-up or analysis of individual shots, biosensors are used as technologically advanced devices both in settings with limited resources and in sophisticated medical settings.
Considering the complex and critical situation in the field of environmental protection, and the state of natural waters from the aspect of pollution with heavy metals, and taking into account the toxicity of heavy metal ions, it is necessary to continuously work on finding new efficient techniques for their detection. Conventional analytical techniques can no longer satisfy the needs of constant monitoring and frequent field analysis of water because they are expensive, often with bulky equipment and a long analysis time, and require well-trained analysts. Biosensors can be used to overcome the limitations of conventional methods. In the future, designing a biosensor with the appropriate material will surely help in the selective identification of metal ions not only from water but also from any other matrix.
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