Provenance of HMAC component materials.
\\n\\n
Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 179 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 252 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\n'}],latestNews:[{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"},{slug:"intechopen-s-chapter-awarded-the-guenther-von-pannewitz-preis-2020-20200715",title:"IntechOpen's Chapter Awarded the Günther-von-Pannewitz-Preis 2020"}]},book:{item:{type:"book",id:"5921",leadTitle:null,fullTitle:"Textiles for Advanced Applications",title:"Textiles for Advanced Applications",subtitle:null,reviewType:"peer-reviewed",abstract:"This book presents a global view of the development and applications of technical textiles with the description of materials, structures, properties, characterizations, functions and relevant production technologies, case studies, challenges, and opportunities. 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It can be more serious than the other forms of skin cancer because it may spread to other parts of the body (metastasize) and cause serious illness and death. For malignant melanomas standard treatment options have remained remarkably static over the past 30 years [1,2]. At present, the incidence of melanoma continues to increase despite public health initiatives that have promoted protection against the sun. Thus, during the past ten years, the incidence and annual mortality of melanoma has increased more rapidly than any other cancer and according to the American Cancer Society estimate, there will have been approximately 76,250 new cases of invasive melanoma diagnosed in 2012 in the United States, which resulted in approximately 9,180 deaths [3].
Unfortunately, the increase in incidence has not been paralleled by the development of new therapeutic agents with a significant impact on survival. Although many patients with melanoma localized to the skin are cured by surgical excision, increased time to diagnosis is associated with higher stage of disease, and those with regional lymphatic or metastatic disease respond poorly to conventional radiation and chemotherapy with 5-year survival rates ranging from 10 to 50% [4]. Currently, limited therapeutic options exist for patients with metastatic melanomas, and all standard combinations currently used in metastasis therapy have low efficacy and poor response rates. For instance, the only approved chemotherapy for metastatic melanoma, dacarbacine, has a response rate of about 10% and a median survival of 8-9 months. The other approved agent for advanced melanoma is high dose interleukin-2, which can induce dramatic complete and durable responses [2]. However, only one patient in twenty derives lasting benefit. These data indicate the needed for alternative therapies for this disease and recent results indicated that combined therapies could became an attractive strategy to fight melanoma [2].
Other example of the complications involved in melanoma chemotherapy is the limited effectiveness of antifolates. Although methotrexate (MTX), the most frequently used antifolate, is an efficient drug for several types of cancer, it is not active against melanoma [5-7]. Undoubtedly, unravelling the mechanisms of melanoma resistance to MTX could yield important information on how to circumvent this resistance and could have important pharmacological implications for the design of novel combined therapies. Thus, although an old drug, MTX could become a valuable tool with which to improve melanoma therapy.
The antifolate methotrexate was rationally-designed nearly 70 years ago to potently block the folate-dependent enzyme dihydrofolate reductase (DHFR). DHFR (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) catalyses the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) in the presence of coenzyme NADPH as follows: DHF + NADPH + H+ → THF + NADP+. This enzyme is necessary for maintaining intracellular pools of THF and its derivatives which are essential cofactors in one-carbon metabolism. Coupled with thymidylate synthase (TS) [8], it is directly involved in thymidylate (dTMP) production through a de novo pathway. DHFR is therefore pivotal in providing purines and pyrimidine precursors for the biosynthesis of DNA, RNA and amino acids. In addition, it is the target enzyme [9] for antifolate drugs such as the antineoplastic drug MTX and the antibacterial drug trimethoprim (TMP). The mechanisms of resistance to MTX have been extensively studied, mainly in experimental tumours propagated in vitro and in vivo [5,10,11]; however, the specific basis for the resistance of melanoma cells to MTX is unclear. During decades the mechanism of resistance of melanoma to MTX was associated with general mechanisms of resistance detected in other epithelial cancer cell including reduced cellular uptake of this drug, high intracellular levels of DHFR and/or insufficient rate of MTX polyglutamylation, which diminishes long-chain MTX polyglutamates from being preferentially retained intracellularly [11]. However, recently, a melanoma-specific mechanism of resistance to cytotoxic drugs, including MTX, has been described [6,12,13].
Antifolate resistance in cancer cells is believed to be a multifactorial process in which dysregulation of apoptosis, insufficient rates of MTX polyglutamylation, and enhanced DNA repair play important roles [11,14]. In melanoma, another classical mechanism of resistance to MTX, the upregulation of endogenous dihydrofolate reductase (DHFR) activity, has been described [5]; however, the contribution of this mechanism to the overall resistance of melanoma to MTX as well as its possible impact on DNA damage response pathways in cells is unknown. ‘Thymineless’ death, which occurs upon the depletion of cellular dTTP pools, has been proposed as a mechanism by which antifolate drugs promote apoptosis in cancer cells [15,16]. Although the mechanism of dTTP depletion-induced apoptosis is yet to be determined, Pardee’s group recently postulated that dTTP controls E2F1, which regulates both DNA synthesis and apoptosis. This hypothesis was based on the observation that MTX increased E2F1 levels in sensitive cancer cells, resulting in an increase in the E2F1-mediated apoptotic cascade.
Eukaryotic cells have developed complex checkpoint pathways that monitor DNA for damage or incomplete replication. Checkpoint pathways are amplified upon detection of aberrant DNA structures and lead to a delay in cell cycle progression during which damage can be repaired or replication be completed. Alternatively, in case of heavily damaged or seriously deregulated cells, checkpoint activation can result in apoptosis. As such, checkpoint mechanisms are essential for the maintenance of genomic integrity [17]. When vertebrate cells experience replication arrest or undergo DNA damage by UV irradiation, the ATR kinase [ataxia telangiectasia mutated (ATM)- and Rad3-related kinase] phosphorylates and activates the Chk1 protein kinase. Activated Chk1 inhibits Cdc25 phosphatases, which control inhibitory phosphorylation sites on cyclin-dependent kinases, the latter being critical regulators of cell cycle transitions [18,19]. Because the ability of cells to delay cell cycle progression and halt DNA synthesis represents a defensive mechanism that spares potential toxicity [20], the activation of Chk1 by MTX could constitute a key event in the resistance of melanoma to MTX.
In addition to these cellular mechanisms of resistance to MTX in melanoma, other mechanism that includes liver transformation of the drug has also been reported. A paradoxical response of malignant melanoma to MTX in vivo and in vitro has been described [21]. The authors observed that MTX showed consistent cytotoxicity for melanoma cells in vitro but was ineffective at equivalent concentrations in vivo. MTX undergoes oxidation to its primary metabolite 7-hydroxy-MTX (7-OH-MTX) in the liver by the enzyme aldehyde oxidase [11] and therefore, this transformation has been proposed as a novel mechanism of resistance to explain this paradox [11,21]. In contrast to the large body of literature available on the multiple modalities of MTX resistance, very little is known regarding the ability of 7-OH-MTX to provoke antifolate-resistance phenomena that may disrupt MTX activity. Recent studies seem to indicate that 7-OH-MTX which exceeds by far MTX in the plasma of MTX-treated patients can provoke distinct modalities of antifolate-resistance that severely compromise the efficacy of the parent drug MTX [22].
Experiments from our laboratory and others provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export [6,12,13]. Concretely, we have described that folate receptor α (FRα)-endocytotic transport of MTX facilitates drug melanosomal sequestration and cellular exportation in melanoma cells, which ensures reduced accumulation of MTX in intracellular compartments [6]. An important observation in this study was that MTX was a cytostatic agent on melanoma cells. These cells were resistant to MTX-induced apoptosis but responded to the drug by arresting their growth. A similar response was observed when the murine B16/F10 melanoma cell line was grown in low folate. After 3 days in folate-deficient medium the cells had restricted proliferative activity and also increased their metastatic potential [23]. Taking this into consideration, the results indicate that MTX might also induce depletion of intracellular reduced folate coenzymes by reducing their transport though the FRα and/or competing with them for the reduced folate carrier (RFC). Melanoma cells may be highly sensitive to intracellular depletion of folate coenzymes, and in this situation may enter into a “latent” state. This form of melanoma should indeed be highly resistant to MTX, since antifolate drugs are more effective on fast-dividing cells, which require continuous DNA synthesis. Most likely, the high increases of DHFR expression in cells treated with MTX [5] would represent an adaptation mechanism that allows cells to survive with low intracellular concentrations of folate coenzymes. Increasing the recycling of folate molecules the cells would maintain other cellular functions that are dependent on folate coenzymes, such as the synthesis of purines, pyrimidines, amino acids and methylation reactions. The presence of this “latent” form of melanoma should be critical for the resistance to MTX during in vivo therapies. Although MTX chemotherapy could initially halt the development of the tumor, after clearance of the drug from the body the melanoma cells may reinitiate their progression, possibly with an increased metastatic potential [23].
A defect in intracellular folate retention is another recognized mechanism of drug resistance [5,10,11,21]. In addition to a decrease in antifolate polyglutamylation, melanoma cells may also export cytotoxic drugs by melanosome sequestration [12]. The results presented in this study indicated that drug exportation was an operative mechanism of resistance to MTX in melanoma cells. Although the mechanism by which cytotoxic drugs are sequestered into melanosomes remains unclear, we demonstrated that MTX-melanosome trapping may be a consequence of its FRα-endosomal transport [6]. To test the importance of this process on the resistance of melanoma to antifolates, we silenced the expression of the melanosomal structural protein gp100/Pmel17, which is known to play a critical role in melanosome biogenesis [24]. Recently, Xie and collaborators [13] provided the first direct evidence that disruption of the process of normal melanosome biogenesis, by mutation of gp100/Pmel17, increased sensitivity to cisplatin. We also observed that effective silencing of gp100/Pmel17 significantly increased the sensitivity of melanoma cells to MTX, favouring MTX-induced apoptosis. This observation strongly supports the hypothesis which indicates that melanosome biogenesis is a specialization of the endocytic pathway [25,26]; however, the exact mechanism by which MTX induces abnormal trafficking of early endosomes in melanoma cells, favoring the exportation of melanosomes, is still unclear. Whether MTX blocks the formation of carrier vesicles operating between early and late endosomes, inhibits the delivery of endocytosed material from endosomes to lysosomes, promoting, thus, the generation of exosomes [26] and/or induces a failure of lysosomal acidification, which is essential for normal endocytosis [27], remains to be determined.
A) Possible mechanisms for transport and trafficking of folates in melanoma cells. (B) Mechanisms to explain the MTX-induced depletion of DHF in melanoma cells. (C) Folate deficiency induces DHF depletion and enhances the transactivational potential of E2F1. (D) Excess of dTTP inhibits E2F1-mediated apoptosis and activates Chk1 in melanoma cells. High levels of DHFR and TS could reactivate de novo dTMP biosynthesis impeding depletion of dTTP. Excess of dTTP would prevent apoptosis by several mechanisms. First, dTTP is an allosteric inhibitor of ribonucleotide reductase (RR), the enzyme which reduces cytidine diphosphate (CDP) and uridine diphosphate to dCDP and dUDP.
To explore the relationship between MTX exportation and melanosome trafficking, we studied the possible interaction of MTX with melanin [6]. Such interaction was confirmed by incubating this drug with synthetic 3,4-dihydroxyphenylalanine (DOPA)-melanin. Importantly, folic acid and 5-methyl-THF (5-MTHF), the natural source of cellular folates, did not appear to interact with synthetic DOPA-melanin. A comparison of the interaction of several folates (folic acid and 5-MTHF) and antifolates (MTX and aminopterin) with synthetic DOPA-melanin indicated that the double amino group of the pterin ring is an important molecular requirement for the drug-melanin interaction. Therefore, the physiological importance of the high affinity of melanin for antifolates, such as MTX and aminopterin, for drug melanosomal sequestration is also another important issue that remains to be addressed. Endocytic transport of molecules involves several processes, including the fusion of early and late endosomes and the dissociation of receptor-ligand complexes through the acidic pH of preformed vesicles [28]. After melanosome biogenesis from MTX-loaded endosomes, dissociated MTX could be trapped in the melanosomes by its interaction with melanins. In contrast, folate substrates would not be sequestered in melanosomes due to their low affinities for melanin; facilitated by the acidic pH of this organelle, uncharged reduced folates would leave the melanosome by passive diffusion and reach the cytosol, where they would become available for cellular functions. Therefore, elucidation of the molecular basis for the (anti)folate interaction with melanins could have important therapeutic implications, and this study might be used as a guide for the synthesis of new antifolates or for using existing antifolates in ways that escape melanin trapping.
Although MTX is exported within a few hours in contact with cells, in this short time, MTX is capable of inducing important changes in folate metabolism by depleting dihydrofolate (DHF) early on and by inducing the expression of folate-dependent enzymes later on [7]. The increased expression of DHFR is a common occurrence in melanoma and other cancer cells in response to MTX treatment; however, the observed depletion of DHF was completely unexpected. The pathways that comprise folate-mediated one-carbon metabolism have been suggested to function in a metabolic network that interconnects the three biosynthetic pathways, namely de novo purine biosynthesis, de novo dTMP biosynthesis, and homocysteine remethylation. Recent studies provide direct evidence for cell cycle–dependent nuclear dTMP biosynthesis in the nucleus [29]. However, there are many unanswered questions regarding the role and regulation of nuclear de novo dTMP biosynthesis. Nothing is known about the transport, processing, and accumulation of folates into the nucleus, the one-carbon forms of folate present in the nucleus, and the relationship between cell cycle dependency of de novo dTMP biosynthesis and cell cycle-dependent accumulation of nuclear folate [29]. Although there is no data of how the homocysteine remethylation cycle is compartmentalized, the observation that MTX affected both DHF synthesis and E2F1 methylation (see below) seem to indicate that both the de novo dTMP biosynthesis and the homocysteine remethylation cycles might operate simultaneously in the nucleus.
Using HeLa and MCF-7 cells, Stover and coworkers observed that cytoplasmic serine hydroxymethyltransferase (SMTH), TS, and DHFR are all translocated into the nucleus during S and G2/M phases following their modification by the small ubiquitin-like modifier (SUMO) [30,31]. This finding indicated that the folate cycle may be compartmentalized and that dTMP and DHF synthesis may occur in the nucleus during DNA synthesis. In a recent study, Wollack et al. [32] characterized 5-MTHF uptake and metabolism by primary rat choroid plexus epithelial cells in vitro. They distinguish two different processes for 5-MTHF transport, one that was FRα dependent and the other that was independent of this receptor and mediated by the proton couple folate transporter or reduced folate carrier (RFC). This investigation revealed that cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. Thus, 5-MTHF taken up via a non-FRα–mediated process was rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remained non-metabolized and associated to endocytic compartments. The observation that MTX induces the overall depletion of FRα in melanoma cells [6] would suggest that MTX might also induce depletion of reduced folate coenzymes associated to endocytic compartments (Figure 1A and 1B). Therefore, a possible explanation for the depletion of DHF during MTX exposure could be that this drug diminishes the required supply of folates to the nucleus for the maintenance of both dTMP and DHF synthesis; however, how melanoma cells can control endocytic pathways to supply their own nucleus with folates is unknown. Recent studies have indicated that some endocytic proteins are also involved in direct signaling pathways from membranes to the nucleus, and mechanisms for the nuclear translocation of intact or fragmented endosome-localized proteins have been identified [33]. Another possibility is the existence of a late endosome-lysosome transport mechanism for folate [34]. The proximity of lysosomes to the nucleus suggests that folates could be released into the perinuclear region of the cytoplasm, perhaps facilitating their nuclear entry during cell division following the disassembly of the nuclear membrane [29].
Although the uptake of 5-MTHF into mammalian cells is mainly mediated by the RFC, the expression of FRα in several epithelial tissues and especially its overexpression in cancerous cells indicate that this receptor may confer a growth advantage to these cells [35]. The high affinity of FRα for 5-MTHF suggest that this GPI-anchored receptor may play an important role in maintaining nuclear folates even at low extracellular concentrations of this vitamin. This hypothesis is supported by the finding that induction of FRα expression in cells that normally do not express this receptor allows the cells to grow in low nanomolar folate concentrations [36]. On the other hand, the observation that methionine synthase was localized in the nucleus of melanoma cells could explain many of the unanswered questions on the role and regulation of the folate metabolism in the nucleus of these cancer cells. The methionine synthase -mediated catalysis of 5-MTHF would first supply THF and methionine to maintain both dTTP synthesis and the methylation reactions in the nucleus of the cells (Figure 1C) and second would prevent the nuclear accumulation of 5-MTHF, a potent inhibitor of SHMT [29]. Therefore, in melanoma, the existence of a specific folate transport pathway from the plasma membrane to the nucleus, mediated by FRα, is possible [37] and could shed light on the unknown function of overexpressed FRα in cancer cells [38].
MTX acts as a cytostatic agent in melanoma cells [6]. To discriminate between the mechanisms by which MTX could induce cell growth arrest without inducing apoptosis, the effect of this drug on the cell cycle of several melanoma cell lines was analysed [7]. The results indicated that, in all the tested melanoma cell lines, MTX conferred an arrest in early S phase; the G1 peak shifted toward the G1/S border, and cells were arrested with a minimal increase in their DNA content. Because S phase arrest has been recognized as a major mechanism of resistance in response to non-toxic concentrations of drugs that induce DNA replication stress, these preliminary results suggest that moderate DNA damage could be responsible for the cytostatic effect of MTX on melanoma cells.
MTX enhances the transactivation potential of E2F1 in melanoma cells. (A) The time-dependent effect of MTX treatment (1 µM) on the expression of E2F1, DHFR, and TS proteins as assayed by western blot (WB). (B) ChIP experiments showing the occupancy of E2F1 and Rb on the DHFR promoter of B16/F10 melanoma cells (*P < 0.05). (C) The upper panels represent the time-dependent effects of MTX (1 μM) treatment on the expression and phosphorylation state of the Rb protein as assayed by WB. The lower panel depicts the Rb mRNA expression as assayed by qRT-PCR. The changes observed after MTX treatment were not statistically significant. (D) Co-immunoprecipitation assays were performed to test the interaction between Rb and E2F1.
To understand the mechanisms involved in G1 cell cycle progression in MTX-treated melanoma cells, the effect of this drug on several G1 cell cycle components was analysed. Although the protein levels of E2F1 were not affected by MTX (Figure 2A), this drug significantly increased the protein levels of DHFR and thymidylate synthase (TS), two E2F1-target genes involved in folate metabolism and required for G1 progression and DNA synthesis (Figure 2A). Chromatin immunoprecipitation (ChIP) experiments that were designed to analyze the occupancy of E2F1 on the DHFR promoter of B16/F10 melanoma cells indicated that MTX stimulated the transcriptional activity of E2F1 (Figure 2B). First, we observed that MTX induced a transient decrease in the hypophosphorylated Rb protein in melanoma cells (Figure 2C) as evidenced by a noticeable lack of Rb co-immunoprecipitation with E2F1 in 10 h MTX-treated SK-MEL-28 cells when compared to untreated controls (Figure 2D). In addition, mass peptide analysis of immunoprecipitated E2F1, after trypsin digestion (Figure 3), indicated that MTX promoted the demethylation of E2F1 at Lys185 (Figures 3B and 3D). A negative crosstalk between methylation and other posttranslational modifications of E2F1, such as acetylation and phosphorylation, has been recently described [39]. We observed that MTX induced the transient co-immunoprecipitation of E2F1 with p300/CBP-associated factor (P/CAF) (Figure 3B), an interaction that has been associated with the transcriptionally active hyperacetylated form of this transcription factor [40]. The hyperacetylated status of E2F1 after MTX treatment was also confirmed by MALDI-TOF mass spectrometry (Figures 3B and 3D). In response to severe DNA damage, the E2F1 protein is stabilized through distinct mechanisms, including direct phosphorylation by Chk2 at Ser364 [41] or ATM kinase at Ser31 [42]. As we did not observe phosphorylation of E2F1 after MTX treatment (Figures 3C and 3D), these data further suggest that MTX induced moderate DNA damage without inducing double strand breaks (DSBs) [43].
MTX increased E2F1 levels in sensitive cancer cells [16]. However, we did not observe an MTX-mediated increase in E2F1 levels in melanoma cells (Figure 2A) [7], a result that could be explained, at least in part, by the results obtained after determination of dNTP pools in melanoma cells (Figure 4). Contrary to the effects of MTX in most cancer cells [16], this drug increased the levels of dTTP in melanoma. Increased levels of dTTP were accompanied by a decrease in dCTP levels, which resulted in a nucleotide imbalance that favored thymidine excess. The MTX-induced expression of DHFR and TS (Figure 2A) and the low levels of MTX accumulated in melanoma cells [6] could explain this paradoxical response of melanoma cells to a cytotoxic drug that typically depletes dTTP levels.
The data obtained in our study indicate that melanoma cells respond to the lack of folate coenzymes by enhancing the transactivational potential of E2F1. We observed that treatment of melanoma cells with MTX transiently affected the stability of Rb and the posttranslational state of E2F1 [7]. A crosstalk between the methylated and acetylated forms of E2F1 has been suggested [39]. Methylated E2F1 is prone to ubiquitination and degradation, whereas the demethylation of E2F1 favors its P/CAF-dependent acetylation. Together, the results suggest a model whereby the MTX-induced degradation of Rb and the demethylation of E2F1 would result in the accumulation of E2F1 in its \'free\' state, and in the absence of DNA damage, free E2F1 would be acetylated, leading to the transcription of genes required for S phase (Figure 1C). The activation of E2F1 by MTX would allow S phase transition in melanoma cells, and importantly for melanoma survival, cells would recover an operative folate cycle, thereby restoring the original status of the Rb/E2F1 system. In the absence of exported MTX, high levels of TS and DHFR would impede the lethal depletion of dTTP and in turn, would produce a nucleotide imbalance that would favor a dTTP excess. Contrary to thymidine depletion, excess thymidine stops cells in S phase by blocking synthesis of DNA, an effect known as ‘thymidine block’ (Figure 1D) [15]. Recently, a mechanism by which dTTP allosterically feedback controls E2F1 has been proposed [15,16]. According to this mechanism, excess of dTTP inhibits E2F1 accumulation acting either upon production of E2F1 or its degradation. Because control of E2F1 is essential for cell survival, this mechanism would prevent E2F1 accumulation, which would result in activation of apoptosis through a process that involves p53 or p73, cytochrome c, and caspases (Figure 1D) [44].
MTX induces demethylation and hyperacetylation of E2F1 in melanoma cells. (A) Schematic representation of the E2F1 protein. Residues susceptible to methylation (K185), acetylation (K117, K120, and K125), and phosphorylation (S31 and S364) are shown. (B) Relative intensity of unmethylated [(K)NHIQWLGSHTTVGVGGR(L); m/z 1820.0229] and hyperacetylated [(R)HPGKAcGVKAcSPGEKAcSR(Y); m/z 1589.8399] peptides in E2F1-trypsin digested samples. Peptides were analyzed in untreated SK-MEL-28 cells (CN) or treated for 10 h with 1 μM MTX (*P < 0.05). Intensities were normalized with respect to an internal matrix control. (C) Cell lysates from SK-MEL-28 cells that had been treated with 1 μM MTX were used for IP assays with E2F1 to test the co-immunoprecipitation of E2F1 with P/CAF and the phosphorylated state of E2F1. (D) MALDI-TOF mass spectra of tryptic digests of immunoprecipitated E2F1. The characteristics peptides involving posttranslational modifications of E2F1 (methylation = Me, acetylation = Ac, and phosphorylation = P), as well as their measured and theoretical m/z are shown.
MTX does not deplete dTTP levels in melanoma cells. dNTP quantification in SK-MEL-28 control cells and cells subjected to MTX (1 μM) treatment (*P < 0.05). Data collected from the left panel was used to determine the total amounts of each dNTP at each time point. The percent contribution of each dNTP to the total pool after 24 h of treatment is represented.
Excess thymidine induces little detectable DNA damage in the form of DSBs. The ATR-mediated response appears to play a more prominent role under these cellular conditions [45]. As it is known that the central mechanism responsible for Chk1 activation upon DNA damage is the distribution of ATR into nuclear foci [46], the effects of MTX on the localization of ATR and the phosphorylation of Chk1 at Ser345 were analyzed by confocal microscopy and western blot, respectively (Figures 5A and 5B). Time- and dose-dependent experiments clearly indicated that MTX induced Chk1 phosphorylation in melanoma cells. Because Chk1 phosphorylation may not directly correspond to Chk1 activation, we next analyzed the dose-dependent effects of MTX on the stability of Cdc25A (Figure 5B). We found that Chk1 phosphorylation led to a corresponding decrease in Cdc25A abundance, indicating that MTX not only conferred Chk1 phosphorylation, but it also activated Chk1. Conversely, phosphorylation of Chk2 was not observed in melanoma cells that had been treated with MTX for as long as 48 h (Figure 5B), indicating that this drug specifically induced Chk1 activation in response to DNA single strand breaks (SSBs). To determine the extent to which Chk1 activation affected the resistance of melanoma to MTX, we took two independent experimental approaches. First, we silenced the expression of Chk1 in SK-MEL-28 (p53 mutant) cells and studied the sensitivity of the cells to MTX (Figure 5C). The results indicated that the downregulation of Chk1 increased the sensitivity of SK-MEL-28 cells to MTX and led to apoptosis. As a second approach, we evaluated the ability of Chk1 to protect B16/F10 murine cells (p53 wild-type) from MTX-induced apoptosis by first inducing an S phase arrest with MTX and then treating the S-arrested cells with a combination of MTX and 7-hydroxystaurosporine (UCN-01). We observed that B16/F10 S phase-arrested cells were sensitive to MTX treatment after the effective inhibition of Chk1 (Figure 5C).
MTX activates Chk1 in melanoma cells. (A) SK-MEL-28 cells were treated with 1 μM MTX for 24 h and then examined for ATR nuclear foci. Nuclei were stained with DAPI. (B) The dose-dependent effects of MTX on Chk1 phosphorylation and Cdc25A degradation in SK-MEL-28 after 24 h of drug exposure (*P < 0.05). MTX (1 μM) induced the time-dependent phosphorylation of Chk1, but not Chk2, in different melanoma cell lines. (C) Chk1 siRNA sensitizes SK-MEL-28 cells to MTX-induced toxicity (left panel). siControl (siCN)- and siChk1-transfected cells were treated with increasing doses of MTX for 48 h (*P < 0.05). The effective silencing of Chk1 was tested by WB. The induction of the phosphorylated form of Chk1 was analyzed after 24 h of MTX treatment (1 μM). The induction of apoptosis by UCN-01 in MTX-arrested B16/F10 cells is shown in the right-side panel. Cells were incubated with 1 μM MTX continuously for 32 h, and 50 nM UCN-01 was added at 24 h to one group of cells following splitting of the culture. As a control experiment, SK-MEL-28 cells were treated with 50 nM UCN-01 only for 32 h.
Inhibitors of DNA synthesis, such as excess thymidine, hydroxyurea, and camptothecin, are normally poor inducers of apoptosis; however, these agents become potent inducers of death in S phase cells upon the small interfering RNA-mediated depletion of Chk1 [45]. Here, we observed that MTX activated Chk1 and induced an early S phase arrest in melanoma cells lines that were harboring either wild-type or mutant p53. The impact of MTX on the survival of Chk1-silenced melanoma cells and cells co-treated with UCN-01 indicates that MTX provokes a ‘thymidine block’-like effect and that S phase arrest, as a result of Chk1 activation, might constitute a major and general p53-independent mechanism that is responsible for the resistance of melanomas to MTX. However, it would be difficult to understand this extreme resistance without taking into account the melanosome-mediated exportation of MTX. The activation of the DNA damage response pathway reflects the magnitude and extent of DNA damage that occurs in response to a specific genotoxic agent, and a dual role of Chk1, depending on the extent of DNA damage, has been proposed [45]. Thus, Chk1 may play an anti-apoptotic role in response to weaker replication fork stresses, whereas more catastrophic damage, such as the accumulation of DNA strand breaks, may result in the activation of apoptosis by Chk1. Together, the results indicate that low intracellular levels of MTX in melanoma induce moderate DNA damage that favors the anti-apoptotic role of Chk1 (Figure 1D).
Although melanoma resistance to MTX was initially thought to be due to the classical mechanisms of resistance that have been observed in other epithelial cells, recent discoveries indicate that the resistance of melanoma to MTX might be due to the idiosyncrasies of these cancer cells [6,12] where drug melanosomal sequestration and its subsequent cellular exportation may have a marked protagonist. Unravelling the mechanisms of melanoma resistance to MTX could, therefore, yield important information on how to circumvent this resistance and could have important pharmacological implications for the design of novel combined therapies. Taking into account these observations, uses of combined treatments with MTX, to prevent melanosomal drug sequestration [6,12] or to avoid MTX-induced S phase arrest [19], are rational therapeutical approaches. The observation that MTX induces cellular depletion of DHF in melanoma [7] could generate novel combined therapies to efficiently inhibit DHFR with antifolates transported into the cells by FRα-independent processes. Also, of great interest is the observed effect of MTX on the posttranslational status of E2F1 in melanoma (Figure 3). Various studies have suggested that E2F1 plays dual roles in cell survival/apoptosis [47-50]. Therefore, the MTX-induced demethylation and acetylation of E2F1 could favour melanoma cell death when combined with E2F1-stabilizing drugs. In addition to E2F1 phosphorylation, acetylation has also been recognized to play a role in the activation and stabilization of the E2F1 protein during DNA damage and apoptosis [40]. A possible strategy to favour E2F1 apoptosis in melanoma by the combination of MTX with E2F1-stabilizing drugs is depicted in Figure 6.
Proposed mechanism for the regulation of E2F1 by MTX. E2F1 is regulated by its interaction with Rb and by several posttranslational modifications, including methylation (Me), acetylation (Ac) and phosphorylation (P) [39]. The effects of MTX (red dashed line) on E2F1 status and that result in melanoma resistance are shown. A possible strategy to stabilize E2F1 (green dashed lines) to induce apoptosis in melanoma cells is also displayed.
Melanoma, the most aggressive form of skin cancer, is notoriously resistant to all current modalities of cancer therapy, including to the drug MTX. Melanosomal sequestration and cellular exportation of methotrexate have been proposed to be important melanoma-specific mechanisms that contribute to the resistance of melanoma to methotrexate. In addition, other mechanisms of resistance that are present in most epithelial cancer cells are also operative in melanoma. This chapter reviews how melanoma orchestrates these mechanisms to become extremely resistant to methotrexate, where both E2F1 and Chk1, two molecules with dual roles in survival/apoptosis, play prominent roles. The results indicated that MTX induced the depletion of DHF in melanoma cells, which stimulated the transcriptional activity of E2F1. The elevate expression of DHFR and TS, two E2F1-target genes involved in folate metabolism and required for G1 progression, favoured dTTP accumulation, which promoted DNA single strand breaks and the subsequent activation of Chk1. Under these conditions, melanoma cells are protected from apoptosis by arresting their cell cycle in S phase. Excess of dTTP could also inhibit E2F1-mediated apoptosis in melanoma cells. In addition, these discoveries could open the way for the development of new combined and directed therapies against this elusive skin pathology.
Industries annually generate millions of metric tons of solid by-products, and most of these materials have been landfilled at considerable cost since. Modern society has been developing beneficial reuse of industrial by-products in a variety of applications [1, 2, 3]. Recycling of waste construction materials saves natural resources, saves energy, reduces solid waste, reduces air and water pollutants and reduces greenhouse gases [4, 5]. The transportation, construction and environmental industries have the greatest potential for reuse because they use vast quantities of earthen materials annually. Replacement of natural soils, aggregates and cements with solid industrial by-products is highly desirable [1, 2].
The steel industry produces a myriad of metal components for industrial chains such as the automobile industry, which in turn generates mineral discarded sand moulds (waste foundry sand/WFS) that end up occupying large volumes in landfills [6]. The major portion of the WFS is considered as non-hazardous waste and is currently deposited in a special WFS landfill that is remote from areas of settlement [7, 8, 9, 10].
The metal casting industry annually discards about 10% of foundry sand for production, i.e. approximately an estimated 9–10 million tons of WFS each year, in the USA [5, 10, 11]. Generally speaking, approximately 1 ton of foundry sand is needed to produce 1 ton of metal casting [8, 12]. WFS can be used as an alternative material (fine aggregate in asphalt mixtures) in highway constructions allowing the increasing of the lifespan of landfills [13].
This work analyses the physical and mechanical behaviour of asphalt mixtures, using the WFS as a mineral filler in asphalt concrete, in 5% (in mass) of maximum replacement to conventional Portland cement (CP). The waste was obtained from an industry located in the free-trade zone of Manaus city, Amazon State, Brazil. The results showed that the addition of industrial WFS in asphalt mixture resulted in adequate performance of the mixtures.
Waste foundry sand is generated by industries that use sands, binders and additives to form moulds and cores for castings. Sands are chosen for several reasons; they are readily available everywhere, inexpensive, highly refractory and readily bonded by clays or other inorganic and organic materials [8, 9, 14]. The mould forms the outside of the castings; the core forms the internal shape. When the part to be made has deep recesses or hollow portions, sand cores must be provided in the mould [3]. The material to be used to form moulds and cores in a foundry should have cohesiveness and porosity properties at the same time. Adding binder (bentonite, resins, cement, sodium silicate and oils) will improve the cohesiveness of the sand grains but will tend to reduce porosity. Additives are those materials which are added to the bonded sands to improve properties, either during the moulding process or during the casting process or both [8]. The moulding processes which involve sand are (1) green sand moulding (or clay-bonded sand, [12]), (2) chemically bonded process and (3) shell moulding process [3, 5, 8, 9]. The most commonly used process is green sand moulding [15]. Green sand is composed of four major materials. Sand comprises 85–95% of the green sand mixture. Most often the sand is inert silica, but olivine and zircon sand are also used [8, 15, 16, 17]. Approximately, 4–10% of the mixture is made of some form of clay, e.g. bentonite. The clay acts as a binder for the green sand and provides strength and plasticity. Combustible additives like sea coal, cereal, fuel oil and wood flour typically make up from 2 to 10% of the green sand mixture. The final additive of green sand is water which is usually added in small percentages (2–5% by weight) [5, 8]. Chemically bonded sands are those that use furan, phenolic urethane and acid cured no-bake systems, as well as alkyd and phenolic urethane cold box processes. Shell moulding uses a mixture of sand and thermosetting resin (usually phenol formaldehyde) to form the mould [8, 17].
The physical, chemical and mechanical characteristics of virgin sand make it a popular material for construction engineering, but after several reuses in moulds and cores, it becomes WFS [7]. The grain size distribution of WFS is quite uniform, with a majority of the sizes (85–95%) falling within a narrow range between 0.6 and 0.15 mm, and 5–12% is smaller than 0.075 mm [5, 8, 10] or between 1 and 16.5% [14]. According to Tikalsky et al. [17], more than 80% of the particles by mass are concentrated by size between 0.15 and 0.70 mm, compared to 0.30–4.75 mm for conventional fine aggregate. Most of the WFS materials reported are found to be medium to fine sand. WFS have been found to be too fine to satisfy the specifications for general fine aggregate [8, 10, 12]. WFS has uniform equidimensional subangular to rounded grains, and a few has rounded grains [8, 10, 17, 18].
For density and unit weight, the values found for the WFS were very close to conventional aggregate [13]. The bulk specific gravities reported in the literature on WFS ranged from 1.985 to 2.722 [8, 17]. In most of the cases WFS have been reported to be almost dry. The moisture content as received for WFS were reported to be in the range of 0.0–4.85% [8, 17, 18]. Concerning absorption, the values are relatively higher than those obtained for the natural aggregate, due to the presence of organic matter [6, 8]. The percentage absorption values on WFS samples have been reported to vary between 0.3 and 6.2% [3, 8, 17].
Over the past three decades, there have been several studies around the world on the use of WFS in engineering works, in different areas: base and subbase layers of highway construction [19, 20, 21], embankments [22, 23], hydraulic barriers [24], asphalt mixtures [3, 7, 16, 25, 26], etc.
Highway subbase layers using WFS have been shown to resist winter conditions (freeze–thaw cycles) better than specimens of reference materials [5, 17, 19]. If a subbase layer stabilised with WFS is compacted in field at dry of optimum content then it will have an increase in its strength [19, 20, 27].
It has been mentioned in the literature that the fines of WFS affect the properties of asphalt concrete negatively [7, 28]. The amount of WFS used in an asphalt mixture depends largely on the amount of fines in the WFS [5, 12, 14, 29]. Studies have recommended that WFS should replace successfully as much as 15% (in mass) of the conventional sand (fine) content in asphalt concrete [3, 9]; 8–10, 10–20 and 10%, respectively, in engineering practice in Pennsylvania, Michigan and Tennessee States [5]; 35% [30]; 15% [26]; 10% [7, 13]; 15% [10, 31]; 35% [27, 32]; and 15–30% [14].
Concerning physical characteristics, the densities of the mixtures decreased as the percentage of WFS in the asphalt concrete increased [7, 9, 10, 12, 13, 17, 32]. Percentage of air voids and voids in the mineral aggregate (VMA) were found to increase with blending of increased quantities of WFS [8, 9]. The optimum asphalt content (4.9–6.8%) for HMA mixtures containing various amounts of foundry sand is comparable to the content of mixes not containing foundry sand [14, 17]. The OAC increases with increase in the WFS percentage [13], although Miller et al. [14] found lower values for mixtures containing WFS, in relation to control ones. According to this author, the mixtures obtain the higher percentage of OAC with the WFS with the higher amount of particles passing the #200 sieve. This happens due to the fineness properties of material and increase of surface area [10, 32].
Regarding the mechanical characteristics, the Marshall stability of the asphalt concrete samples containing WFS decreases as the quantity of WFS is increased [3, 6, 7, 8, 10, 12, 29, 32]. The flow values of mixtures decreased with increasing percentage of WFS in the asphalt concrete mixtures [7, 8, 9, 10, 13]. The indirect tensile strengths of the asphalt cement mixtures decreased as the percentage of WFS material was increased [7, 8, 9, 10, 12, 13, 32]. However, Abdulsattar and Mohammed [25] found that all the WFS mixtures that they analysed showed higher tensile strength than the control mixture. According to Tikalsky et al. [17], the level of air voids and saturation greatly influenced the indirect tension values.
In relation to moisture susceptibility, WFS has little effect on top-down fatigue cracking resistance and moisture susceptibility of the mixtures [32]. When WFS replacement is higher than 15%, asphalt mix may become more sensitive to moisture damage (i.e. stripping) due to the presence of silica [10, 27]. WFS, on average, decreases the unconditioned tensile strength and thus the durability of asphalt mixtures; on the other hand, WFS do not necessarily increase or decrease a mixture’s rutting potential but do improve fatigue performance [17].
The experimental procedure of this research contemplates the dosage and physical and mechanical tests on five hot-mixed asphalt concrete (HMAC) mixtures using the conventional Portland cement filler (as reference) and four other mixtures using WFS, replacing the cement gradually in proportions of 25%. This residue was produced by the foundry industrial process of a company located in free-trade zone of Manaus city, Amazon State, Brazil, which produces clutch assembly lines (pressure and friction plates, discs, outer housing, etc.) for the motorcycle industry. Figure 1a shows one of the several kinds of pieces that are produced in that industry, while Figure 1b presents the WFS studied. The annual production of WFS in that industry was about 1500 tons in 2014 (SUFRAMA, 2016). The coarse aggregate (natural pebble) came from the “Japurá” River (an Amazon River affluent) riverbeds and was extracted by dredging, but it was acquired in the local market. The fine aggregate (clean sand) came from mining extraction in the vicinity of the city (about 30–50 km), but it was acquired in the local market as well. The mineral filler used was Portland cement II-Z-32 type. Finally, asphalt cement (AC) 50/70 grading was used, produced by the oil refinery of Manaus (REMAN). The materials used in this research and their respective origins are listed in Table 1.
(a) A piece (to be deburred) produced at the trade zone of Manaus city industry. (b) WFS to be tested.
Material | Origin |
---|---|
Sand | Market of Manaus |
Pebble | Market of Manaus |
Portland cement (PC) II-Z-32 (mineral filler) | Market of Manaus |
Asphalt cement (AC) (50/70 grading) | Oil refinery of Manaus (REMAN) |
Waste foundry sand (WFS) | Industry of free-trade zone of Manaus |
Provenance of HMAC component materials.
All mineral aggregates used in the asphalt mixtures were tested according to the standards described in Table 2, mainly by the Brazilian highway standards, which are most similar to known international standards. In relation to the asphalt cement (AC—50/70 penetrating grading), it was submitted to complete characterisation according to standards shown in Table 3.
Material | Brazilian standard | Title | Acceptance parameters (Brazilian standard) | Similar international standard |
---|---|---|---|---|
Pebble | NBR NM 53/2009 | Coarse aggregate—determination of the bulk specific gravity, apparent specific gravity and water absorption | Greater than 0.88 and 2.00 g/cm3; less than 18%, respectively | ASTM-T-85 |
Pebble | NBR NM 51/2001 | Coarse aggregate—test method for resistance to degradation by Los Angeles machine | Less than 50% | AASHTO-T-96 |
Pebble, Sand, Fillers | NBR NM 248/2003 | Aggregates—sieve analysis of fine and coarse aggregates | Within granulometric range | ASTM-C136/C136M-14 |
Pebble | NBR 12583/1992 | Coarse aggregate—coating to bituminous binder | Qualitative test (visual analysis) | — |
Sand | NBR NM 52/2009 | Fine aggregate—determination of the bulk specific gravity and apparent specific gravity | Greater than 1.60 and 2.60 g/cm3, respectively | ASTM-C128–01 |
Fillers | NBR NM 23/2001 | Portland cement and other powdered materials—determination of density | Greater than 3.00 g/cm3 | ASTM-C188–09 |
WFS | NBR 16137/2010 | Non-destructive testing—material identification by spot test, X-ray fluorescence spectrometry and optical emission spectrometry | — | ASTM-C114–15 |
WFS | — | Wavelength dispersive X-ray fluorescence spectrometry | — | ASTM-C1365 |
Aggregate characterisation tests.
Brazilian standard | Test | Unity | Similar international standard | AC 50/75 |
---|---|---|---|---|
NBR 14756 | Apparent specific gravity, 25°C | g/cm3 | AASHTO T 228 | 1.010 |
NBR 6576 | Penetration, 25°C, 100 g, 5 s | 0.1 mm | AASHTO T 49 | 58 |
NBR 14950 | SSF viscosity, 135°C | s | AASHTO T 72 | 160 |
NBR 15184 | Brookfield viscosity, 135°C, sp21, rpm 20 | cP | AASHTO T 316 | 286 |
NBR 6560 | Softening point | °C | AASHTO T 53 | 53 |
NBR 6293 | Ductility, 25°C | cm | AASHTO T 51 | >120 |
Properties of asphalt cement (AC—50/70 penetrating grading) used in the mixtures.
In order to avoid the presence of impurities, the residue was washed in sieves Nos. 200, 300 and 400, before subjected to characterisation tests and used in asphalt mixtures. The WFS filler was subjected to chemical analysis (XRF) made by an X-Ray spectrometer equipment (720 energy dispersive, Shimadzu), through drying and subsequently pressing the sample in a disc form. The equipment can perform analyses from sodium to uranium, has a rhodium tube and is cooling by liquid nitrogen. Besides that, the WFS filler was also submitted to the X-Ray diffraction (XRD) in order to be characterised its crystalline phases. The equipment used in the analysis was the D8 Focus-Bruker diffractometer, with monochromatic cuprum radiation (CuKα, λ = 1.5418 Å), operating at 35 kV and 40 mA. A laser particle size analyser was used to determine with precision the particle size of both mineral fillers (PC and WFS).
Since the tests were performed 10 years ago, asphalt concrete studies were developed through the traditional Marshall method and not by current Superior Performing Asphalt Pavements (Superpave) methodology. After the characterisation of all components of asphalt concrete, the materials were classified in the “C” granulometric range limits of Brazilian highway specifications following the Marshall dosage method, as shown in Figure 2. The curves obtained fitted in the area defined by the two curve limits of the “C” range, minimum and maximum. After fixing the particle size distribution of aggregates of the mixture, the probable optimum asphalt content (OAC) was estimated by the expression derived from the work of Duriez (1950) based on the specific surface of the aggregates:
XRD analysis for WFS filler.
where S is the specific surface area of aggregate (m2/kg), G is the percentage retained on sieve 9.5 mm, g is the percentage passing on sieve #9.5 mm e retained on sieve 4.8 mm, A is the percentage passing on sieve #4.8 mm e retained on sieve 0.3 mm, a is the percentage passing on sieve #0.3 mm e retained on sieve 0.074 mm and f is the percentage passing on sieve 0.074 mm.
Then, the probable OAC was calculated, using the following expression:
where Tca is the OAC in relation to the mass of the aggregates (%) and m is the richness modulus of AC, varying from 3.75 (wearing course with high stiffness) to 4.00 (wearing course with low stiffness).
If the mean bulk specific gravity of the total aggregate is less than 2.60 or greater than 2.70, then the content obtained in the previous item should be corrected by the following expression:
where T′ca is the corrected OAC in relation to the mass of the aggregates (%) and δam is the mean bulk specific gravity of the total aggregate.
Finally, the OAC is calculated in relation to the entire mixture:
where Pca is the final value of OAC in relation to the total mixture (%).
From that OAC value were adopted two points below it (each 0.5%) and two points above it (each 0.5%).
Five HMAC mixtures were analysed whose grain size proportions are shown inTable 4. The mixture 1 was used as reference, for 100% of Portland cement as mineral filler. The other mixtures used WFS as mineral filler, replacing Portland cement in gradual proportions each 25%. At the end, the results were compared between the mixtures with and without WFS according to the physical and mechanical tests performed.
Oxide | SiO2 | Al2O3 | SO3 | Fe2O3 |
---|---|---|---|---|
Content (%) | 93.68 | 3.97 | 1.66 | 0.41 |
Composition of oxides present in WFS filler.
The experimental procedures were defined as follows, for each mixture [33]: (i) determination of the AC working temperatures from Saybolt-Furol viscosity test in the range of 85 ± 10 and 140 ± 15 SSF for mixing and compaction, respectively; (ii) the components (aggregates + AC) were mixed at a temperature of 146°C for approximately 2 min; (iii) the mix was placed in the Marshall mould and compacted mechanically with 75 blows on each side of the specimen; (iv) the specimen were left at rest for 24 h at room temperature; (v) after that, the specimens were left in a water bath at 60°C for 2 h; (vi) finally, they were placed in the compression mould and submitted to compression in order to determine the rupture load and flow value. Thus, all physical and mechanical parameters of HMAC mixtures were determined by the Marshall method.
From Eq. 4, an initial OAC value of 6.15% for mixture 1 was adopted, with m = 3.75. Nevertheless, the mixture showed excessive fluid, with AC in excess. Hence, OAC = 4.5% was considered. It is noteworthy that three specimens were cast for each AC content to find the final OAC of each the mixture (mixtures 1–5), whose range varied from 3.5 to 5.5%, at each interval of 0.5%. Figure 3a presents the results of OAC for each mixture.
Marshall physical and mechanical characteristics of studied mixtures: (a) optimum asphalt content, (b) bulk specific gravity, (c) air void volume, (d) asphalt-void ratio, (e) Marshall stability and (f) flow value.
After the tests, the Marshall parameters of the mixtures were determined: bulk specific gravity (BSG), theoretical maximum specific gravity (TMG), air void volume (AVV), voids in the mineral aggregate (VMA), voids filled with asphalt (VFA), asphalt-void ratio (AVR), Marshall stability (STA) and flow value (FLV). The optimum contents of AC adopted were those with an AVV value of 4%.
Three samples with cylindrical forms were moulded for the determination of the static indirect tensile strength (ITS) by diametrical compression for each type of mixture, at each OAC. The ITS individual value was obtained through the expression
where σt is the individual static ITS (kPa), T is the static rupture load (kN), r is the sample radius (m) and h is the sample height (m).
Three samples were moulded for determining the resilient modulus (RM) of each mixture. This mixture was then placed in the mould and compacted mechanically with 75 blows on each side of the sample. Then, the specimen were submitted to a repeatedly vertical compression load F at a maximum stress level less than or equal to 20% of the ITS. The RM adopted was the arithmetical mean value determined at 300, 400 and 500 load application F.
Hence, the value of the RM was determined by the expression [33]
where RM is the individual resilient modulus (MPa), F is the cyclic vertical load diametrically applied on specimen (N), δ is the elastic strain recorded for 200, 400 and 500 load applications (mm), h is the sample height (mm) and μ is Poisson’s ratio.
The fatigue test was performed to define the number of loading repetitions as a function of controlled stresses in diametrical compression samples with the load applied at a frequency of 1 Hz, with 0.10 s of repeated loading duration through the same resilient modulus equipment, increasing in tensile strain until the specimen is completely disrupted at a constant temperature of 25°C. The fatigue curve was determined in seven stress levels (7.5, 10, 15, 20, 25, 30 and 40% of the static ITS) with two specimens per level. The fatigue resistance was evaluated according to the fatigue curves generated by testing, which introduces the relationship between fatigue strength and fatigue life. The fatigue equation in this study was calculated using the formula given in the following equation [34]:
where Nf is the fatigue life (in cycles) and σf is the fatigue stress (MPa), i.e. the tension stress applied during the test. The equation provides a linear relationship between them using a bilogarithmic scale, in which “n” is the gradient and “k” is the intercept.
The study of permanent deformation was made using the static creep test applying a static and continuous compression load on a specimen moulded according to the Marshall methodology. The specimen was placed in the axial position and then was subjected to an applied tension of 0.1 MPa, distributed over the entire contact surface of the specimen for a period of 60 min at a temperature of 40°C. The permanent deformations were measured continuously along that time, and then the specimen was discharged, waiting for 15 min for the stabilisation of the viscous deformations, which were measured continuously too. The total strain (Dt) after the recovery period can be obtained as:
where Δh75 is the specimen height change after the final recovery period, i.e. 75 min after the start of the test load (mm), and ho is the specimen initial height taken in the axial direction of loading (mm). Table 5 shows the mechanical tests performed on HMAC mixtures, while Figure 4 presents all tests carried out on components and mixtures.
Brazilian standard | Title | Acceptance parameter (Brazilian standard) | Similar international standard |
---|---|---|---|
DNER-ME 043/1995 | Asphalt mixtures—Marshall test | OAC ≥ 6% STA ≥ 5 kN 3% < AVV < 5% | ASTM D5581-07a |
NBR 16018/2011 | Asphalt mixture—stiffness determination by repeated load indirect tension test | — | ASTM D4123–82 |
NBR 15087/2012 | Asphalt mixtures—determination of tensile strength by diametrical compression | ≥0.65 MPa | ASTM D 6931–17 |
DNER-ME (provisional standard)/2017 | Hot-mixed asphalt concrete—fatigue under repeated loading, constant tension, using the indirect tension test | — | FHWA-Protocol P07/2001 |
— | Standard test methods for tensile, compressive and flexural creep and creep rupture of plastics | Dt ≤ 0.02 mm/mm in 75 min | ASTM D 2990–09 |
Mechanical characterisation tests carried out on HMAC mixtures.
Flowchart of the laboratory tests.
Figure 2 indicates the result of XRD analysis for WFS filler. As shown in the figure, WFS is essentially formed by quartz mineral, as expected. Table 4 shows the composition of the main oxides present in the WFS filler obtained by XRF analysis. The high percentage of silica confirms the XRD analysis of the material [8, 16, 17].Table 6 indicates the physical characteristics of the aggregates. WFS aggregate apparent specific gravity of WFS is very close to conventional aggregates (pebble and sand) [7, 9, 10, 13] each other except for PC. Pebble had a Los Angeles abrasion loss below the maximum allowed by the Brazilian standard, which is 50%. The WFS had 76.25% of its particle sizes passing at #200 sieve and are slightly larger than that of Portland cement, i.e. it is too fine to replace part of the fine aggregate of the asphalt mixes [8, 10, 12], thus demonstrating that the residue could only replace part or total filler fraction.
Aggregate | Apparent specific gravity (g/cm3) | Absorption (%) | Los Angeles abrasion loss (%) | d90 (mm) | d50 (mm) | d10 (mm) |
---|---|---|---|---|---|---|
Pebble | 2.66 | 1.92 | 40.0 | 12.0 | 7.0 | 2.5 |
Sand | 2.63 | — | — | 1.5 | 0.35 | 0.12 |
Filler (PC) | 3.03 | — | — | 0.063 | 0.020 | 0.004 |
Filler (WFS) | 2.65 | — | — | 0.133 | 0.040 | 0.004 |
Physical characteristics of aggregates.
Notes: d90, d50 and d10 are the particle size for which 90, 50 and 10% of the all particles, in mass, are finer than it.
Table 7 shows the resulting granulometric composition of the mineral aggregates with and without the addition of WFS. It is observed that all the mixtures were composed with the same amount of aggregates, varying only the proportion between the two types of the filler fraction. Conventional mixture 1 used PC exclusively, while mixture 2 used WFS as filler exclusively. The other mixtures had variations between permutations of PC and WFS proportions. The grain size distribution of the mineral aggregates, the “C” range maximum and minimum limits of the Brazilian highway specification and the resulting aggregates of mixtures 1 and 2 are shown in Figure 5.
Aggregate | Mixture designation | ||||
---|---|---|---|---|---|
1 (%) | 2 (%) | 3 (%) | 4 (%) | 5 (%) | |
Pebble | 62.0 | 62.0 | 62.0 | 62.0 | 62.0 |
Sand | 33.0 | 33.0 | 33.0 | 33.0 | 33.0 |
Filler (cement) | 5.0 | 0.0 | 3.75 | 2.5 | 1.25 |
Filler (WFS) | 0.0 | 5.0 | 1.250 | 2.5 | 3.75 |
% Total | 100.0 | 100.0 | 100.0 | 100.0 | 100.0 |
Granulometric composition of mineral aggregate mixtures with and without WFS addition.
Grain size distribution and limit curves of mineral aggregates.
Figure 3 shows the main physical parameters of the mixtures, obtained through the Marshall methodology. OAC values of the mixtures containing WFS are comparable to the control in mixture 1 [14, 17]. Mixture 1 obtained the lowest OAC (4.5%), whereas mixtures with WFS had little bit higher OAC values, whose contents increased as WFS proportions were increased too [13]. This reason probably is due to the absorption characteristics of this residue, and not due to the grain size [10, 32], since CP has larger particle size and therefore smaller surface area and thus should consume less AC, at the same proportion of WFS.
It was observed that all five mixtures met the Brazilian standards regarding the physical Marshall parameters (OAC, AVV, VMA and AVR). Mixture 1 had a higher GMB values than all other mixtures with WFS and was therefore the densest. The other mixtures maintained a slight decrease of this parameter, when the proportion of WFS in the mixture was increased [7, 9, 13]. Mixture 2 (100% WFS filler) had the highest amount of AVV and the second largest AVR among all mixtures. AVV values increased when WFS content were increased in the mixtures [8, 9, 10, 32].
High amounts of AVV and AVR tend to negatively influence STA and FLV values, given the viscous characteristic of AC. Thus, mixture 1 showed the best performance, with the highest STA and lowest FLV values. Among the mixtures using WFS, mixture 5 (one fourth WFS + three fourths PC) was the one that presented the highest value of GMB, thus being the densest, and also presented the highest value of STA; however, it had the highest FLV value too. The FLV values of the WFS blends were higher than the PC blends, which characterises a higher AC consumption of these blends. In summary, the use of WFS decreased the stability of blends [6, 8, 10, 12, 32] while increasing their fluency. This latter is in disagreement with that observed by the author cited previously. Even so, all mixtures showed STA values higher than the minimum required (>5 kN).
There was a certain tendency that static ITS values will decrease as WFS content increased [7, 9, 10, 13, 32]. Mixtures 3 and 5 presented higher values of this parameter than control mixture 1 [25]. All asphalt mixtures presented values above the minimum value of the Brazilian standard (>0.65 MPa). This is a good indication for durability of the mixtures since fatigue life is a function of ITS. There was not an apparent correlation between AVV and static ITS values (Figure 6).
Mechanical characteristics of studied mixtures: (a) static indirect tensile strength, (b) resilient modulus, (c) RM/ITS ratio and (d) total strain (static creep).
The use of WFS decreased the RM values. Mixture 1 presented the highest value, followed by mixture 2. In Brazil, the relationship between RM and static ITS (RM/ITS) has been used as an analysis parameter to evaluate the behaviour of asphalt mixtures related to fatigue life. As a rule, mixtures with RM/ITS ratio around 3000 exhibit good structural behaviour because they allow the use of thinner asphalt wearing layers for the same fatigue life; that is, they characterise mixtures that are not susceptible to early development of permanent deformations because they are not rigid enough. In this sense, mixture 3 was the only one that met this criterion. On the other hand, the conventional mixture 1 presented the highest value of this ratio, thus indicating a more rigid behaviour.
Figure 7 shows the comparison between asphalt mixtures in relation to the stress-controlled fatigue test. For the acquisition of fatigue curves, the average value of the RM and the static ITS of each mixture were used. Between Mixtures 1 and 4, the best-fitting straight lines were very close to each other, with a parallelism between the line slopes, and both mixtures can be considered to have practically the same fatigue life. Mixture 2 presented the shortest fatigue life, while mixture 5 presented the longest fatigue life, standing out among the others. For applied stress differences up to 0.4 MPa, Mixtures 1, 2 and 4 behave similarly.
Fatigue life for the mixtures with and without WFS content.
It should be noted that mixture 5 presented the second best ITS result and the second closest value of the RM/ITS ratio around 3000, thus justifying the use of this parameter as a quantitative indicator of fatigue life of asphalt mixtures. The fatigue life test on mixture 3 was not performed.
Regarding the permanent deformation, Mixtures 2 and 3 presented lower values than mixture 1, while mixture 5 presented the highest value among the others. There was no direct relationship with AVV, since, of all of them, mixture 5 presented the lowest value of voids. Mixture 3 presented the lowest value of permanent deformation, confirming again the good indicative of the RM/ITS ratio around 3000 in predicting the behaviour of asphalt mixtures for fatigue and permanent deformations. All mixtures presented permanent deformation values below the conventional criterion of 0.020 mm/mm and do not have the tendency to be susceptible to premature permanent deformations.
This work analysed five asphalt mixtures, one using 100% CP as a filler and the other four using WFS, with a maximum proportion of 5% (by weight) of the total aggregate. The WFS residue used consisted of almost 94% silica, without organic compounds, with apparent specific gravity similar to clean sand and slightly coarser than CP.
All mixtures with WFS residue presented physical and mechanical parameters within the Brazilian standards, following the Marshall methodology, although with lower STA and higher FLV values. The use of WFS increased static ITS values, while decreased MR values. The mixtures with WFS showed total permanent deformation values less than 2% after 75 min of the test. The RM/ITS ratio around 3000 proved to be a good indication of mixtures with better performance against fatigue life and permanent deformation.
Finally, the use of WFS as a mineral filer in asphalt mixtures proved to be adequate, meeting the criteria of Brazilian standards in physical and mechanical tests.
The authors would like to thank Prof. Dr. Laura Maria Goretti da Mota, from Pavement Laboratory of COPPE/Federal University of Rio de Janeiro (UFRJ), for some laboratorial tests carried out in that place. This work was supported by the CNPq [grant number 620244/2008-9]; FAPEAM [scholarship].
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\n\n*These prices do not include Value-Added Tax (VAT). Residents of European Union countries need to add VAT based on the specific rate in their country of residence. Institutions and companies registered as VAT taxable entities in their own EU member state will not pay VAT as long as provision of the VAT registration number is made during the application process. This is made possible by the EU reverse charge method.
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