Proteomics in Biomarkers of Chemical Toxicity

C H A P T E R

65 Proteomics in Biomarkers of Chemical Toxicity Christina Wilson-Frank Purdue University, College of Veterinary Medicine, Animal Disease Diagnostic Laboratory, Department of Comparative Pathobiology, West Lafayette, IN, United States

INTRODUCTION Toxicoproteomics is emerging as an important tool in elucidating biochemical pathways of chemical toxicity and identifying potential biomarkers for establishing risk assessment, level of concern, and early detection of toxicant exposure. Proteomic analyses allow global simultaneous, high-throughput monitoring of proteins following exposure to chemical toxicants and provides an effective strategy to identifying useful biomarkers indicative of toxicity. One of the major challenges in proteomics research, particularly when probing biological samples for biomarkers of chemical toxicity, is the inherent complexity of the proteome. However, advancements in sample preparation (to reduce sample complexity), analytical instrumentation, and automated sequence database searching have enabled for simplified and improved, highthroughput analysis and more accurate protein identification. When evaluating the proteome in complex biological samples, the proteomics platforms commonly employed include a technology that globally separates proteins, combined with the use of mass spectrometry for protein identification. To achieve global separation of proteins from complex samples, gel-based technologies are more commonly used in proteomics studies for chemical toxicity, such as 1-dimensional gel electrophoresis (1DE), 2-dimensional gel electrophoresis (2-DE), and 2dimensional-difference gel electrophoresis (2-DGE). Recently, advances in analytical instrumentation have increased the separating capacity and resolution of chromatographic systems; therefore, the use of highperformance liquid chromatography (HPLC) and ultrahigh-performance liquid chromatography are emerging as popular platforms for high-throughput, complex protein separations. The most common analytical platforms used for protein sequencing and identification

Biomarkers in Toxicology, Second Edition https://doi.org/10.1016/B978-0-12-814655-2.00065-7

are matrix-assisted laser desorption/time-of-flight mass spectrometry (MALDI-TOF MS) and tandem mass spectrometry (MS/MS) which can include time-of-flight (TOF), quadrupole (Q), or ion trap mass spectrometry. Using these technologies, researchers have been able to measure qualitative and quantitative changes in proteins in samples such as serum/plasma, blood, brain, liver, and kidney samples to distinguish unique biomarkers of chemical toxicity. Much of the proteomics research conducted to elucidate candidate biomarkers of chemical toxicity predominantly involves recognizing quantitative changes in proteins after biological systems or cells are subjected to acute or chronic exposure to toxicants. However, an alternative approach to defining potential, candidate biomarkers of toxicity involves using protein network building tools, such as MetaCore or Ingenuity Pathways Analysis software (Wang et al., 2013; Huang and Huang, 2011). Proteins that are differentially expressed in chemical toxicity studies can be subject to protein interaction network analysis, which predicts proteineprotein interactions and highlights which molecular pathways in which those proteins play a key role. Not only does this highlight probable molecular mechanisms of toxicity but also may allow investigators to focus on a specific group of proteins or protein in that pathway to evaluate as prospective biomarkers. Pesticides, herbicides, heavy metals, and most organic contaminants are well-studied chemical toxicants that are known to be potential occupational or environmental health hazards. This chapter describes proteomics investigations conducted in these biological samples that identify potential, predictive biomarkers of chemical toxicity and lead to an improved understanding of mechanisms of toxicity of these chemicals. The potential biomarkers identified in these investigations have been reported to be a result of either cause acute or chronic chemical toxicity and have been

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recognized as environmental contaminants or have a high risk of occupational exposure.

HEAVY METALS Arsenic Arsenic is a carcinogenic metalloid and is recognized as one of the most toxic heavy metals (Tokar et al., 2013). Environmental exposure to arsenic primarily occurs via arsenic-contaminated drinking water, because trivalent and pentavalent forms of the heavy metal are widely distributed in the environment. Because arsenicals are used in the smelting industry and in the manufacturing of pesticides, herbicides, and products used in wood preservation, occupational exposure to arsenic is also a concern (Tokar et al., 2013). Individuals exposed to arsenic are at risk for developing cancer, hyperkeratosis, cardiovascular disease, and kidney and liver damage. For most of these cases, chronic exposure to arsenic is implicated. Due to the lack of adequate biomarkers to assess exposure to arsenic in these cases, various proteomics methodologies have been used to help reveal candidate biomarkers for early diagnosis of arsenicinduced diseases. Arsenic-induced hyperkeratosis has been associated with skin cancer and other internal cancers and is endemic in parts of the world in which individuals are chronically exposed to arsenic in drinking water (Hong et al., 2017; Hsu et al., 2013). To identify useful biomarkers for early diagnosis of arsenic-induced, hyperkeratosis in exposed individuals, a proteomic study was conducted using skin samples from the palms and feet of individuals who had skin lesions who resided in an arsenic-contaminated area in Shanyin, China (Guo et al., 2016). One protein, DSG1 (desmoglein 1), was significantly suppressed in the skin from arsenicexposed individuals. DSG1 is a cadherin-like protein that promotes anchoring of the keratin cytoskeleton to the cell membrane; therefore, suppression of this protein can result in abnormal epidermal cell differentiation. Proteins that were present in increased concentrations in affected individuals were FABP5 (fatty acid-binding protein 5) and KRT6C (keratin 6C). KRT6C and other keratins are responsible for maintaining epidermal cell-to-cell adhesion, and mutations in these proteins have been linked to other types of skin abnormalities (Guo et al., 2016). FABP5 plays an integral role in fatty acid metabolism and is known to be upregulated in various forms of human cancers, possibly promoting tumor development (Chen et al., 2011; Levi et al., 2013; Ogawa et al., 2008). Cardiotoxicity can also occur with chronic arsenic exposure, as it causes cardiac arrhythmias and can alter myocardial depolarization (Chen and Karagas, 2013). To

evaluate the mechanism of arsenic cardiotoxicity, rats were given sodium arsenate and changes in the proteomic profiles of heart tissue from treated rats were evaluated (Huang et al., 2017). Eighty-one proteins changed with arsenic exposure and 14 of the proteins were associated with cardiovascular function and development. Proteins that were upregulated included cardiac troponin T and creatinine kinase. Cardiac troponin T is present in cardiomyocytes and is responsible for myocardial contractility. It is a known, specific biomarker for acute cardiotoxicity and concentrations will increase after myocardial injury. Other proteins involved in cardiac contractility were also identified and were downregulated with arsenic exposure. These proteins include myosins, tropomyosin alpha-1 chain, and galectin-1. Other proteins identified that may serve as potential biomarkers include talin 1 and vinculin, both cytoskeletal proteins important for myocardium morphogenesis, and vimentin (filament protein important for enhancing protective effects on myocardial ischemic injury).

Cadmium In addition to occurring naturally in the environment, cadmium is also a major environmental contaminant due to being widely used in the production of textiles, plastics, batteries, and fertilizers (Hulla, 2014). Because tobacco plants are known to accumulate cadmium from the soil, individuals smoking tobacco are known to have increased levels of cadmium in their blood and kidneys (Satarug and Moore, 2004). Prolonged exposure to cadmium can lead to accumulation in the proximal tubule epithelial cells and glomerulus, ultimately leading to nephrotoxicity (Pari et al., 2007). Measuring blood urea nitrogen and creatinine are the traditional biomarkers used to assess kidney damage; however, when high levels of these biomarkers are detected, significant kidney damage has already occurred. In an effort to identify early biomarkers of nephrotoxicity, researchers have used proteomics to investigate the effects of cadmium on renal cells. In one study using 2D-gel electrophoresis/MALDI-TOF MS and SILAC/LC-MS, HK-2 human kidney epithelial cells were treated with cadmium chloride (Kim et al., 2015). Of the several proteins identified in this study, HSPA8 (heat shock protein) and ENO1 (alpha-enolase) increased in the treated cells. The increase in ENO1 and HSPA8 was suggested to be due to cadmium-induced cell death through oxidative stress. Another study investigating cadmium nephrotoxicity in human renal cells identified 27 proteins effected by cadmium exposure (Galano et al., 2014). Fifty percent of the proteins identified are involved in apoptosis, whereas others identified were found to be important

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HEAVY METALS

for protein synthesis and cytoskeleton formation. Similar to the previous study, this group of investigators also identified an increase in ENO1. It is important to note that HSPA8 (and other heat shock proteins) and ENO1 are also increased when human kidney cells are damaged by other toxic agents (Kim et al., 2015). Therefore, these proteins may be candidates for biomarkers of early kidney damage as opposed to specific biomarkers for cadmium-induced nephrotoxicity.

Chromium (VI) Hexavalent chromium (Cr (VI)) is a toxic, heavy metal commonly used as an oxidizing agent for stainless steel production, welding, chrome pigment production, and chromium plating (Ashley et al., 2003). Due to its widespread industrial use, concerns regarding environmental contamination and occupational exposure to Cr (VI) have been raised due to its proven toxicity to ecosystems and also because it is a known human carcinogen (Tokar et al., 2013). Recently, investigators have imposed the use of comparative proteomics and serum protein expression profiling to identify biomarkers of occupational exposure and also to gain a better understanding of the mechanism of carcinogenesis of Cr (VI). In an attempt to identify novel serum proteins that change with occupational exposure to Cr (VI), blood samples were collected from 107 chromate workers and were analyzed using nano-flow HPLC/MS and critical proteins of interest that were identified were verified using ELISA (Hu et al., 2017). When compared with the control group, there were 44 differentially expressed serum proteins found in occupational Cr (VI) exposure, all involved in 16 important signaling pathways associated with the immune system, C-reactive protein (CRP), sonic hedgehog protein (SHH), and calcium. Although more studies need to be conducted to garner an understanding of the noted regulatory proteins and mechanisms involved in Cr (VI) exposure, the investigators concluded that CRP and SHH might be potential, novel biomarkers as higher levels of SHH and lower levels of CRP were noted in all of the workers exposed to Cr(VI).

Lead Lead is a major, toxic heavy metal that has been historically used in variety of products including paints, batteries, pesticides, gasoline, and ammunition (Thompson, 2018; Tokar et al., 2013). Although workplace exposure to lead has been progressively reduced, environmental exposure to lead still remains a toxicological concern. Most exposures to lead are through contaminated food or water sources or lead-containing paint chips (Tokar et al., 2013). In mammals, the toxic effect of lead exposure can be widespread ranging from liver or kidney damage

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and neurotoxicity to hematologic effects (Tokar et al., 2013). Proteomic responses to lead neurotoxicity and hepatotoxicity have been conducted that identify possible biomarkers of exposure and also define metabolic pathways involved in lead-induced cell injury. In addition to exposure via inhalation or orally, dermal exposure to lead (e.g., lead acetate) is also a concern (Cohen and Roe, 1991). One group evaluated the hepatotoxic risk caused by derma exposure to lead acetate using 2-DE and MALDI-TOF MS (Fang et al., 2014). In this study, dermal application of lead acetate was applied every 24 h to the back of nude mice and the harvested livers subjected to proteomic analysis and Western blots. The proteins that changed significantly with lead exposure were proteins associated with protein folding, ER stress, apoptosis, and oxidative stress. Specifically, the proteins GRP75 (mortalin) and GRP78 (glucoseregulated protein 78 kDa) were increased by 4.5-fold and 2.0-fold (respectively) with dermal lead exposure. These proteins are responsible for protein folding and targeting misfolded proteins for degradation and also are important indicators of oxidative stress. The investigators suggested that oxidative stress and proapoptotic signals from the ER as a result of lead toxicity were responsible for these proteins being elevated. Other proteins that increased in a dose-dependent manner with lead exposure were ATF6 (activated transcription factor 6), 1RE1a (inositol requiring enzyme), and PERK (protein kinase R-like endoplasmic reticulum kinase). Increased expression of these proteins would lead to cleavage of poly (ADP-ribose) polymerase ultimately resulting in apoptosis of hepatocytes. The investigators also noted that AST and ALT levels were elevated in this study, further confirming liver damage. This study concluded that dermal exposure to lead acetate leads to generation of reactive oxygen species culminating in hepatotoxicosis due to necrosis and apoptosis of hepatocytes. In addition to lead hepatotoxicity, one group investigated the neurotoxic effects of lead exposure by identifying differentially expressed proteins in the hippocampus of juvenile mice given lead acetate in drinking water. Six proteins were upregulated and three proteins were downregulated in lead-exposed mice. Pdhb1 (pyruvate dehydrogenase E1b) and ATPase (proteins involved in cell energy metabolism), Hspd1 and Hspa8 (heat shock proteins involved in cell stress response), and Dpysl2 and Spna2 (proteins involved in protein binding and cytoskeleton development) all increased with oral lead exposure. There were three proteins that decreased with lead neurotoxicity, NADH dehydrogenase, Aars (alanyl tRNA synthetase), and Grb (growth factor receptor protein). The investigators surmised that the increase in ATPase in this study may be compensatory to the decrease in NADH dehydrogenase due to the increased demand for energy by the cells after

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lead toxicity. This group also conducted object recognition tests which revealed that lead administration reduced the memory ability and vertical activity of affected mice to show the biological responses to lead neurotoxicosis induced in these mice.

Mercury Because of its ubiquitous presence in the environment and its propensity to bioaccumulate, mercury poses a significant health hazard to aquatic species and humans. Depending on the form of mercury, it can cause toxicity to the central nervous system, gastrointestinal tract, liver, and kidneys (Gupta et al., 2018a). There have been several studies conducted using proteomics technologies to help elucidate key pathways or biomarkers that may lead to an earlier assessment of exposure risk and to gain an understanding of the mechanism of toxicity of mercury in these organ systems. Proteins involved in mercury neurotoxicity and hepatotoxicity have been studied in medaka fish (Oryzias melastigma). In both studies, chronic exposure to mercury chloride was investigated and potential biomarkers elucidated using 2D-gel electrophoresis/MALDI-TOF MS. Following mercury chloride exposure, there were 33 proteins identified in liver samples from medaka fish of which several proteins significantly changed in the treated fish versus the controls (Wang et al., 2013). Protein markers that increased significantly with mercury exposure included cathepsin D, peroxiredoxin-1 and 2, and natural killer enhancing factor. These proteins are involved in cellular responses to oxidative stress or mediate apoptosis of damaged hepatocytes, highlighting that chronic mercury exposure causes oxidative stress in the liver. Keratin 15 and novel protein similar to vertebrate (PLEC), proteins involved in cytoskeleton assembly, decreased in the treatment group. Mercury has been previously shown to disrupt the cytoskeleton, particularly in cases of acute toxicosis (Wang et al., 2011). Studies investigating the neurotoxicity of mercury in medaka fish revealed similar results. Sixteen proteins were significantly different in the treatment group when compared to controls. As was observed in the chronic hepatotoxicity study, the proteins that changed were important in responses to oxidative stress, cytoskeletal assembly, and metabolic disorders (Wang et al., 2015). The protein markers that increased were cathepsin D, peroxiredoxin-1 and 2, and natural killer enhancing factor. Whereas the proteins that decreased were keratin 15, formimidoyltransferase cyclodeaminase (FTCD), and novel protein similar to vertebrate (PLEC). Novel biomarkers for acute and chronic mercury nephrotoxicity were evaluated in vivo in SpragueeDawley rats orally dosed with mercury chloride and also in vitro in rat

kidney proximal tubular cells (Shin et al., 2017). Proteomic analyses revealed two key biomarkers to be considered for mercury-induced nephrotoxicity. Aldo-keto reductase (AKR7A1) and glutathione-S-transferase (GSTP1) were significantly elevated, in a dosedependent manner, in the kidney and proximal tubular cells. Interestingly, these proteins were notably increased in the absence of detectable increases in BUN and creatinine. AKR7A1 is a protein that is integral in detoxifying metabolites, and GSTP1 is important in protecting cells from oxidative stress. To further corroborate these findings, the investigators were able to show that generation of reactive oxygen species in vitro was increased by mercury in a dose-dependent manner. The authors mention that it is not clear these proteins can serve as noninvasive biomarkers (measured in blood or urine); however, they may serve as biomarker candidates for early stages of nephrotoxicity. Collectively, in the aforementioned proteomic studies, the biomarkers identified for hepatotoxicity, neurotoxicity, and nephrotoxicity allude to mercury causing oxidative stress as a mechanism of toxicity in these organ systems. In addition to tissue proteomics, there has been a study conducted to identify serum biomarkers for organic methylmercury toxicity. One group of researchers dosed mice with organic methylmercury, enriched for serum glycoproteins and analyzed the samples using nano-UPLC/MS/MS (Kim et al., 2013a). Although 21 proteins were differentially expressed in the organic methylmercury-treated mice, two serum protein biomarkers were identified and validated using Western blot analysis and immunohistochemistry. Amyloid P component (SAP) and inter-alpha-trypsin inhibitor heavy chain 4 (ITI-H4) were upregulated. SAP, a protein that exists in amyloid deposits and stabilized chromatin, was the protein that increased the most. ITI-H4 increased more than twofold and is responsible for regulating inflammatory conditions and acute phase response in cells. The authors note that previous proteomic investigations in serum from children with high levels of mercury in their blood (due to eating contaminated fish) also have detectable ITI-H4 (Gump et al., 2012).

PESTICIDES AND HERBICIDES Methyl Parathion Methyl parathion is an organophosphorus pesticide and is classified by the Environmental Protection Agency as a class I toxicant. Due to its widespread use in agriculture, occupational exposure in humans often occurs via inhalation or dermally. Environmental exposure occurs due to contamination of soil and water or due to runoff from sources where methyl parathion

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PESTICIDES AND HERBICIDES

has been applied. The primary mechanism of toxicity of this organophosphate insecticide is through inhibition of acetylcholinesterase in the central nervous system and at the neuromuscular junction (Gupta and Kadel, 1990; Gupta et al., 2018b). Some studies have also shown that methyl parathion can cause DNA damage and induce oxidative stress in a variety of organ systems (Bartoli et al., 1991; Hai et al., 1997). A group of researchers in China have taken an interest in studying changes in cell membrane proteomes to investigate the neurologic and oxidative effects of methyl parathion in brain and other organ systems to establish candidate biomarkers of methyl parathion in those tissues and gain a better understanding of the mechanisms of methyl parathion toxicity. Using 1-DE and 2-DE to separate the membrane proteins and MALDI-TOF MS/MS to sequence the peptides, candidate biomarkers for methyl parathion toxicity were evaluated in zebrafish (Danio rerio) brain and liver, pleural-pedal ganglia from sea slugs (Aplysia juliana), and kidney from scallops (Mizuhopecten yessoensis) (Huang and Huang, 2011, 2012a,b; Chen et al., 2014). The proteins that were identified to be upregulated or downregulated were further validated and confirmed using Western blots and real-time polymerase chain reaction (RT-PCR). Common in all of these proteomic studies, the cell membrane proteins that changed significantly with methyl parathion treatment play a role in oxidative stress, mitochondrial function, energy/cell metabolism, signal transduction, protein synthesis, and degradation and intracellular transport. A list of the membrane proteins that changed with methyl parathion treatment are listed in Table 65.1. The proteins PDIA3 (protein disulfide isomeraseassociated 3), ALDH5A1 (aldehyde dehydrogenase 5A1), and MDH (malate dehydrogenase) were found to have significantly changed in with methyl parathion treatment in zebrafish brain tissue (Huang and Huang, 2011). The investigators suggested that these three proteins are suitable candidate biomarkers as they have been previously reported to be associated with methyl parathion toxicity in other studies (Huang and Huang, 2011). Interestingly, SDH (succinate dehydrogenase) was shown to decrease in sea slug, pleural-pedal ganglia and increase with methyl parathion treatment in zebrafish liver cells (Huang and Huang, 2012a; Chen et al., 2014). Organophosphates have been reported in early studies in aquatic species to decrease SDH activity in tissues, suggesting that these pesticides effect aerobic oxidation in the TCA cycle (Samuel and Sastry, 1989). SDH was also shown to decrease in the pleural-pedal ganglia of sea slug with methyl parathion treatment. However, in zebrafish liver cells, SDH was shown to increase with methyl parathion treatment. The investigators surmised that this increase in SDH might be

TABLE 65.1

Membrane Proteins Altered With Methyl Parathion Treatment

Organ System

Proteins Increased

Proteins Decreased

Braina

GNB1L

GDI2

GNB2

UCHL1

ALDH5A1

Ependymin

PDIA3

STXBP1

DLST IDH3 mMDH EF-Tu b

Pleural-Pedal Ganglia

NADH

SDH

ALD

ICDH

ANN11

MDP

GPCR

TCR

PHB

ILR

b-tubulin

VDAC ABC transporters

Liverc

CD146

TPHR 1

Annexin A2a SDH ACBD5A d

Kidney

ATP synthase

GPx

MAPRE1

MPP

IOX

HPPD PEPCK

a

Huang and Huang (2011). Chen et al. (2014). c Huang and Huang (2012a). d Huang and Huang (2012b). b

compensatory in light of aerobic oxidation being effected in the liver.

Diazinon In addition to acetylcholinesterase inhibition, noncholinergic targets of organophosphate pesticides are also of toxicological concern. Recent studies have suggested that diazinon, an organophosphate pesticide, may cause developmental defects in the absence of acetylcholinesterase inhibition (Harris et al., 2009). To investigate if sublethal doses of diazinon would affect cell development, a targeted proteomic study was conducted in mouse N2a cells to examine if diazinon would induce molecular changes in stress response and axonal cytoskeletal proteins (Harris et al., 2009). Using 2-DE

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and MALDI-TOF MS, 12 proteins involved in cell survival, differentiation, and metabolism changed significantly with diazinon treatment. Notably, the protein cofilin increased approximately fivefold. Cofilin is an actin-binding protein that dissembles actin filaments, suggesting that diazinon disrupts microfilament organization. The researchers concluded that the cytoskeleton may be a target for the neurite inhibitory levels of diazinon, and that proteins important for microfilaments, microtubules, and neurofilaments are affected by diazinon.

Paraquat Paraquat is a potent, restricted, nonselective herbicide that has been associated with high mortality in humans and animals exposed to the product. Ingestion of paraquat can cause multisystem organ failure through production of reactive oxygen species as a result of inhibiting reduction of NADH to NADPH (Gupta, 2018). Paraquat is also selectively taken up into the lungs, causing pulmonary edema, alveolar hemorrhage, and lung fibrosis (Gupta, 2018; Kim et al., 2013c). The use of proteomics in identifying diagnostic biomarkers for pulmonary toxicity in bronchoalveolar lavage fluid has been somewhat successful (Govender et al., 2009). However, isolating biomarkers in noninvasive samples as a result of pulmonary toxicity caused by paraquat intoxication have been challenging. Serum uric acid and acute phase response gene pentraxin-3 have been suggested as prognostic biomarkers for paraquat toxicity in serum (Kim et al., 2011; Yeo et al., 2011); however, these biomarkers may not be ideal for early diagnosis. In an attempt to discover early, diagnostic biomarkers of acute paraquat poisoning, 2-DE and MALDI-TOF MS/MS were used to isolate and identify proteins that change with paraquat poisoning (Kim et al., 2012). In this study, male CD(SD)IGS rats were dosed orally with paraquat dichloride and serum samples were collected from treatment and control groups. Out of approximately 500 protein spots observed, eight proteins were differentially expressed with paraquat treatment. Proteins related to the inflammatory process which included ApoE (apolipoprotein E), Hp (haptoglobin), and C3 (complement component 3) increased in the treatment group. Proteins that decreased with treatment were FGG (fibrinogen g-chain) and Ac-158. Western blot and RT-PCR were used to further validate the usefulness of ApoE, Pphg, and FGG as diagnostic biomarkers of paraquat toxicity. These results concluded that ApoE, Hp, and FGG may be appropriate candidate biomarkers in serum samples for early diagnosis of acute paraquat toxicosis. The increased expression of the protein C3 is interesting in that C3 may play a role in acute inflammatory reactions caused by paraquat

(Sun et al., 2011). Therefore, the investigators concluded that C3 expression in serum may serve as a diagnostic biomarker for paraquat-induced, acute pulmonary inflammation. Proteomic profiling of lung tissue has also been conducted to evaluate changes in proteins expressed in the lung subsequent to paraquat exposure. In this study, protein biomarkers of acute pulmonary toxicosis were investigated using 2-DE and MALDI-TOF MS/MS in rats dosed intraperitoneally with paraquat. It was discovered that CaBP1 (calcium-binding protein 1 regulates calcium-dependent activity in cell cytoskeleton), FKBP4 (important in immunoregulation and protein folding), osteonectin (glycoprotein that binds calcium), and S100A6 (calcium-binding protein that regulates cell cycle progression and differentiation) all increased in rat lung tissue with paraquat treatment.

Glyphosate Glyphosate is widely used as a broad spectrum herbicide and is present in approximately 750 commercial herbicide products (Landrigan and Belpoggi, 2018). The herbicidal mechanism of action is through inhibition of plant enolpyruvylshikimate-3-phosphate synthase, an enzyme involved in the synthesis of tyrosine, tryptophan, and phenylalanine. Without these amino acids, the plant cannot synthesize proteins needed for growth. Due to its widespread use to control broadleaf weeds and grasses, trace amounts of glyphosate can be found in soil, foodstuffs, and water (Landrigan and Belpoggi, 2018). The impact of glyphosate on the environment, particularly in aquatic organisms, is an emerging concern. Glyphosate-based herbicides have been shown to cause a variety of toxic effect in nontarget aquatic organisms, such as hemorrhagic anemia, oxidative stress, and genotoxic effects (Arnett et al., 2014; Rocha et al., 2015). To assess the early toxicological response of glyphosate in fish, guppies (Poecilia reticulata) were housed in aquarium water fortified with 1.82 mg glyphosate/liter and the gill proteome evaluated using 2-DE and MALDI-Q-TOF MS/MS (Rocha et al., 2015). After a 24-h exposure time, 14 proteins involved in energy metabolism, regulation, cytoskeleton maintenance, and stress were identified. Glyphosate exposure inhibited expression of a-enolase, a protein that plays a role in hypoxia tolerance in cells. This inhibition was thought to be due to glyphosate-dependent hypoxia-induced stress in the gills. Proteins important for cytoskeleton regulation were also suppressed in gills from glyphosate-treated fish. Arp4, cortactin-binding protein, myosin-VI-like isoform 4, and actin isoforms decreased with glyphosate exposure, suggesting that glyphosate may interfere with stability of actin filaments in the gills of guppies. The investigators concluded that

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ORGANIC POLLUTANTS

glyphosate modifies expression of gill proteins and promotes changes in the cellular architecture of gill cells in response to hypoxia caused by glyphosate, and that the proteins identified could serve as potential biomarkers for biomonitoring water pollution by herbicides.

ORGANIC POLLUTANTS Benzo(a)pyrene Polycyclic aromatic hydrocarbons (PAHs) are known carcinogens, posing a significant health threat to humans and animals. Their ubiquitous presence in the environment is largely because they are produced by all types of combustion of organic materials such as the incomplete burning of fuels and other substances including tobacco (cigarette smoke) and other plant material (Kim et al., 2013b). One of the most studied PAHs is benzo(a)pyrene, which is metabolized by the cytochrome P450 monooxygenase system and epoxide hydrolase in mammals to form benzo(a)pyrene diol epoxide, a carcinogenic metabolite that forms an adduct with DNA (Di Giulio and Newman, 2013). The genotoxic effects of benzo(a)pyrene have been well studied; however, the effects of benzo(a)pyrene at the protein level are just beginning to be understood. Several researchers have taken advantage of proteomics techniques to begin to evaluate biomarker candidates of benzo(a)pyrene carcinogenicity in bladder cancer, prostate cancer, and lung cancer. Tobacco smoking has been linked to bladder cancer cases in men (Castelao et al., 2001). One concern regarding tobacco is that cigarette smoke has been shown to contain 20e40 ng of benzo(a)pyrene per cigarette (Rodgman et al., 2000). Efforts to understand the possible role of benzo(a)pyrene in bladder cancer development have been attempted using proteomics. In one study, pig primary bladder epithelial cells were exposed to benzo(a)pyrene and the proteome isolated and analyzed using 2-DE and MALDI-TOF MS/MS (Verma et al., 2013). Twenty-five differentially expressed proteins were identified which are known to be important for either mitochondrial repair, DNA repair, or apoptosis. Proteins that were upregulated with benzo(a)pyrene exposure included RAD23, PMSD5, and PMSD4. These proteins play a crucial role in DNA repair and their upregulation suggests that benzo(a)pyrene caused DNA damage to the bladder epithelial cells (Verma et al., 2013). Many of the proteins identified that changed significantly were cathepsin D, VDAC 2 (voltage-dependent anion channel protein), HSP27, and HSP70 (heat shock proteins). These proteins are known to be involved in the mitochondrial death receptor pathway, ultimately inducing apoptosis. These proteins not only may serve as candidate biomarkers for

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benzo(a)pyrene-induced bladder cancer but also this study was able to show that at low concentrations and during short exposure time periods that benzo(a)pyrene can cause DNA damage in bladder epithelial cells leading to cell death due to apoptosis (Verma et al., 2013). Benzo(a)pyrene in cigarette smoke has also been implicated in the development of lung cancer. Evidence has shown that benzo(a)pyrene diol epoxide-DNA adducts occur at the same codon positions that are known to be major, mutational hot spots in human lung cancers (Denissenko et al., 1996). The lack of biomarkers for early diagnosis of lung cancer has been a persistent, clinical challenge. Comparative proteomic analysis of the cellular response to (A549) human airway epithelial cells to benzo(a)pyrene was investigated in hopes of elucidating biomarkers and mechanisms of toxicity (Min et al., 2011). Using 2-DE and MALDI-TOF MS/MS, 23 proteins that play a role in signal transduction, antioxidation, energy and metabolism, and apoptosis were differentially expressed in cell lysates from the airway epithelial cells treated with benzo(a)pyrene. The proteins that increased were annexin A1, thioredoxin, cathepsin D, and heterogeneous nuclear ribonucleoprotein K. Peroxiredoxin I, vimentin, nucleoside diphosphate kinase A, poly(rC)-binding protein 1, and superoxide dismutase (Mn) all decreased in the benzo(a)pyrene-treated cells. Superoxide dismutase (Mn), which is a protein that is considered to be the cell’s first line of defense against superoxides, was very significantly decreased with benzo(a)pyrene exposure in A549 cells (Min et al., 2011). The researchers suggested that accumulation of intracellular reactive oxygen species induced by benzo(a)pyrene exposure is why this protein likely decreased. This proteomic study helped clarify that benzo(a)pyrene can perturb the antioxidant status in airway epithelial cells and highlighted the proteins that change with benzo(a)pyrene toxicity in the lung. In addition to being present in cigarette smoke, benzo(a)pyrene is also known to be present in grilled meat and that consumption of grilled meat has been linked to an increased risk of prostate cancer (Fatma and Kabadayi, 2005). Proteomic investigations conducted to assess benzo(a)pyrene-mediated prostate cancer have been conducted in vitro using PrEC normal prostate epithelial cells (Chaudhary et al., 2006). This investigation revealed 26 proteins that changed with benzo(a) pyrene exposure. Twenty-six proteins were differentially expressed in the prostate epithelial cells with benzo(a)pyrene, most having cellular function in metabolism, signal transduction, cytoskeleton protection, and oxidative stress. Peroxiredoxin I, which increased with benzo(a)pyrene, and peroxiredoxin II, which decreased with benzo(a)pyrene, were of particular interest to the researchers. These proteins are

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thiol-specific antioxidant enzymes and play a role in reducing the oxidative stress in cells (Kang et al., 2005). Peroxiredoxin I expression increase has been shown to increase in several types of cancers, and it is thought that elevation of this protein in cancer is suggestive of the cell’s defense against tumorigenesis (Chaudhary et al., 2006). Peroxiredoxin II suppression can enhance prosurvival pathways through aberrant activation of growth factor receptors resulting in hyperproliferation; therefore, the investigators concluded that decreased peroxiredoxin II may be important for benzo(a)pyrene tumor promotion (Chaudhary et al., 2006). These two proteins may be involved in benzo(a)pyrene toxicity and could potentially serve as biomarkers of benzo(a)pyrene carcinogenicity. Although much of the proteomics studies have been conducted on benzo(a)pyrene, there has been one study investigating plasma protein biomarkers in workers occupationally exposed to mixed PAHs (Kap-Soon et al., 2004). Monitoring occupational exposure to PAHs has historically required measuring for 1hydroxypyrene, a urinary PAH metabolite (Jongeneelen, 2014). However, this marker is not suited for early detection of low-dose, PAH exposure (Kap-Soon et al., 2004). Kap-Soon et al. (2004) analyzed plasma samples from waste gas pollution measurers that worked in an automobile emission inspection center that are exposed to PAHs on a daily basis. To confirm exposure, the 1hydroxypyrene was measured in the workers’ urine and was detected at approximately 4 times higher in the PAH-exposed workers when compared to controls. Proteomic evaluation of the plasma samples from the exposed workers revealed six proteins that increased with PAH exposure which included serum albumin precursor, hemopexin precursor, fibrinogen g-A chain precursor, TCR-b, and CCE channel protein. Serum albumin, hemopexin, and fibrinogen are plasma protein targets of oxidative stress. Their increased presence may be due to attempts to prevent oxidative stress in the cells caused by PAHs (Kap-Soon et al., 2004). TCR-b protein is important for recognizing foreign antigens and subsequently triggering a cascade of signals to activate cells to respond. The authors surmise that the overexpression of TCR-b may be due to this protein recognizing PAH as a foreign antigen and is consequently overexpressed TCR-b (Kap-Soon et al., 2004). CCE channel protein is a plasma membrane protein important for role in calcium hemostasis. The authors suggest that PAH metabolites may target immune cells altering calcium hemostasis leading to apoptosis of cells. In this case, the overexpression of CCE could be due to release from the cell membrane as a result of PAH exposure ultimately perturbing calcium hemostasis and causing

apoptosis. As a result of this study, they concluded that these six proteins could be noninvasive, candidate biomarkers for PAH exposure.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Polychlorinated dioxins, such as TCDD, are very toxic, environmental contaminants that can cause immune system modulation, teratogenesis, and tumor promotion (Sany et al., 2015). TCDD is a by-product in the production of herbicides and can originate from industrial processes such as metal production, fossil fuels/wood combustion, and waste incineration. Similar to benzo(a)pyrene, occupational exposure to TCDD has been reported in individuals working in the waste incineration industry (Phark et al., 2016). Identification of novel biomarkers to use for biomonitoring TCDD exposure in these workers has been made possible through proteomics technologies. In one study, differentially expressed proteins were evaluated and compared in HepG2 cells exposed to TCDD, plasma from rats exposed to TCDD, and plasma from industrial incineration workers exposed to low and high doses of TCDD (Phark et al., 2016). The secreted proteome from the HepG2 cells had seven proteins that increased with TCDD exposure and one protein that decreased. Proteins that increased were GLO 1 (glyoxylase), HGD (homogentisate dioxygenase), peroxiredoxin I, PSMB5 and PSMB6 (proteasome subunit beta type protein), UDP-GlcDH (UDP-glucose-6dehydrogenase), and HADH 9hydroxylacyl-coenzyme A dehydrogenase. STF (serotransferrin) decreased with TCDD treatment. Of these proteins, PSMB5 and peroxiredoxin I were identified in plasma from rats dosed with TCDD and increased in a time-dependent manner in the plasma proteome. GLO 1, HGD, peroxiredoxin I, and PSMB6 were present in the plasma from incineration workers, and protein expression of these proteins was greater in the high-dose exposure group versus the low-dose exposure group. Peroxiredoxin I was present in all three sample models and increased with exposure to TCDD. Interestingly, in proteomic studies exposing PrEC prostate epithelial cells to benzo(a)pyrene to model benzo(a)pyrene-induced prostate cancer, peroxiredoxin I was also overexpressed and increased expression of this protein has been implicated in several types of cancer (Chaudhary et al., 2006). The authors noted that although these proteins may serve as candidate biomarkers for TCDD exposure; more large-scale quantitative studies need to be conducted to further validate their use in biomonitoring occupational exposure to this toxicant.

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CONCLUDING REMARKS AND FUTURE DIRECTIONS Toxicoproteomics applies global protein separation and detection to profile complex biological samples in hopes of identifying candidate biomarkers and gain an understanding of the mechanism of toxicity of the acute and chronic effects of chemical toxicants. Proteomics is now in a unique position to provide meaningful data due to evolving advancements in protein separation and isolation, analytical instrumentation, and database sequence and pathway software programs. However, despite the advances in these technologies, there are still some limitations to these approaches. Future challenges for toxicoproteomics research will require further optimization and validation of candidate biomarkers for chemical toxicity. For example, biomarker peroxiredoxin I was shown to increase in plasma from incineration workers exposed to TCDD, in prostate epithelial cells exposed to benzo(a)pyrene, and in liver cell lysates from fish exposed to mercury (Chaudhary et al., 2006; Wang et al., 2011; Phark et al., 2016). As mentioned earlier, HSP8 (heat shock protein) and ENO1 (a-enolase) were shown to increase in human kidney proximal tubule epithelial cells (HK-2) treated with cadmium in a study evaluating biomarker candidates for cadmiuminduced nephrotoxicity (Kim et al., 2015). HSP8 has also been shown to increase in HK-2 cells exposed to other nephrotoxic agents such as mercury chloride, cisplatin, cyclosporine, and sodium arsenite (Kim et al., 2015). Overall, some of the proteins identified to be candidate biomarkers for specific chemical toxicity may in fact be more appropriate biomarkers for organspecific toxicity. Additionally, although the proteins recognized to change with chemical toxicity may differ, it is evident that these proteins in a few cases are involved in similar cellular responses. For example, across the chemicals reported in this chapter, several implicate differentially expressed proteins involved in oxidative stress response, cell metabolism, and cytoskeleton maintenance (cell structure). Although the proteins associated with these biological processes may differ, it highlights the general mechanisms by which most chemicals exert their toxicity on biological systems. Before differentially expressed proteins will be accepted as reliable biomarkers of chemical toxicity, validation studies on a larger scale will likely be required to ensure the results are repeatable and robust. These studies would also likely require standardization of sample preparation and analysis. Historically, biomarkers for toxicity have included defining one or a few proteins that change with exposure. For example, BUN and creatinine increase with kidney injury; however, many chemicals can cause this

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increase. Further exploiting the global aspect of proteomics techniques, future definition of biomarkers of chemical toxicity may require identifying groups of proteins (e.g., protein profile) that may be related to a specific chemical exposure. Although there were similar proteins that were differentially expressed to comparable degrees in more than one chemical, the entire set of proteins that changed were different when comparing the chemicals evaluated. In addition, some of the researchers conducting the aforementioned proteomics investigations reached for histopathology, RT-PCR, and Western blots to confirm the biomarkers identified in their chemical toxicity studies, further adding creed to the data reported. Ultimately, toxicoproteomics is emerging to be an effective strategy in identifying useful, predictive biomarkers of chemical toxicity.

References Arnett, R., Habibi, H.R., Hontela, A., 2014. Impact of glyphosate and glyphosate-based herbicides on the freshwater environment. J. Appl. Toxicol. 34, 458e479. Ashley, K., Howe, A.M., Demange, M., et al., 2003. Sampling and analysis considerations for the determination of hexavalent chromium in workplace air. J. Environ. Monit. (5), 707e716. Bartoli, S., Bonora, B., Colacci, A., et al., 1991. DNA damaging activity of methyl parathion. Res. Commun. Chem. Pathol. Pharmacol. 71, 209e218. Castelao, J.E., Yuan, J.M., Skipper, P.L., et al., 2001. Gender and smoking-related bladder cancer risk. J. Natl. Cancer Inst. 93 (6), 538e545. Chaudhary, A., Pechan, T., Willett, K.L., 2006. Differential protein expression of peroxiredoxin I and II by benzo(a)pyrene and quercetin treatment in 22Rv1 and PrEC prostate cell lines. Toxicol. Appl. Pharmacol. 220, 197e210. Chen, R., Feng, C., Xu, Y., 2011. Cyclin-dependent kinase-associated protein Cks2 is associated with bladder cancer progression. J. Int. Med. Res. 39, 533e540. Chen, Y., Karagas, M.R., 2013. Arsenic and cardiovascular disease: new evidence from the United States. Ann. Intern. Med. 159, 713e714. Chen, Y., Huang, L., Zhang, Y., et al., 2014. Differential expression profile of membrane proteins in Aplysia pleural-pedal ganglia under the stress of methyl parathion. Environ. Sci. Pollut. Res. 21, 3371e3385. Cohen, A.J., Roe, F.J., 1991. Review of lead toxicology relevant to safety assessment of lead acetate as a hair colouring. Food Chem. Toxicol. 29, 127e139. Di Giulio, R.T., Newman, M.C., 2013. Ecotoxicology. In: Klaasen, C.D. (Ed.), Casarett & Doull’s Toxicology, the Basic Science of Poisons, eighth ed. McGraw- Hill Education, China, pp. 1283e1284. Denissenko, M.F., Pao, A., Pfeifer, G.P., 1996. Preferential formation of benzo(a)pyrene adducts at lung cancer mutational hotspots in P53. Science 274 (5286), 430e432. Fang, J., Wang, P., Huang, C., et al., 2014. Evaluation of the hepatotoxic risk caused by lead acetate via skin exposure using a proteomic approach. Proteomics 14, 2588e2599. Fatma, A.S., Kabadayi, F., 2005. Meat and meat mutagens and risk of prostate cancer in the agricultural health study. Cancer Epidemiol. Biomark. Prev. 17, 80e87. Galano, E., Arciello, A., Piccoli, R., et al., 2014. A proteomic approach to investigate the effects of cadmium and lead on human primary renal cells. Metallomics 6, 587e597.

X. APPLICATIONS OF BIOMARKERS IN TOXICOLOGY

1162

65. PROTEOMICS IN BIOMARKERS OF CHEMICAL TOXICITY

Govender, P., Dunn, M.J., Donnelly, S.C., 2009. Proteomics and the lung: analysis of bronchoalveolar lavage fluid. Proteonomics Clin. Appl. 3, 1044e1051. Gump, B.B., MacKenzie, J.A., Dumas, A.K., et al., 2012. Fish consumption, low-level mercury, lipids, and inflammatory markers in children. Environ. Res. 112, 204e211. Guo, Z., Hu., Q, Tian, J., et al., Proteomic profiling reveals candidate markers for arsenic-induced skin keratosis, Environmental Pollution. 218, 2016, 34-38.Gupta, R.C., Kadel, W.L., 1990. Methyl parathion acute toxicity: prophylaxis and therapy with memantine and atropine. Arch. Int. Pharmacodyn. Ther. 305, 208e221. Gupta, P.K., 2018. Toxicity of herbicides. In: Gupta, R.C. (Ed.), Veterinary Toxicology; Basic and Clinical Principles, third ed. Academic Press/Elsevier, Amsterdam, pp. 495e508. Gupta, R.C., Milatovic, D., Srivastava, A., et al., 2018a. Mercury. In: Gupta, R.C. (Ed.), Veterinary Toxicology; Basic and Clinical Principles, second ed., pp. 573e585. Gupta, R.C., Sachana, M., Mukherjee, I.M., et al., 2018b. Organophosphates and carbamates. In: Gupta, R.C. (Ed.), Veterinary Toxicology; Basic and Clinical Principles, second ed., pp. 573e585. Hai, D.Q., Varga, S.I., Matkovics, B., 1997. Organophosphate effects on antioxidant system in carp (Cyprinus carpio) and catfish (Ictalurus nebulosus). Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 117, 83e88. Harris, W., Sachana, M., Flaskos, J., et al., 2009. Proteomic analysis of differentiating neuroblastoma cells treated with sub-lethal neurite inhibitory concentrations of diazinon: identification of novel biomarkers of effect. Toxicol. Appl. Pharmacol. 240, 159e165. Hong, Y., Ye, B., Kim, Y., et al., 2017. Investigation of health effects according to the exposure of low concentration of arsenic contaminated ground water. Int. J. Environ. Res. Publ. Health 14, 1461e1475. Hsu, L., Chen, G., Lee, C., et al., 2013. Use of arsenic-induced palmoplantar hyperkeratosis and skin cancers to predict risk of subsequent internal malignancy. Am. J. Epidemiol. 177 (3), 202e212. Hu, G., Wang, T., Liu, J., et al., 2017. Serum protein expression profiling and bioinformatics analysis in workers occupationally exposed to chromium (VI). Toxicol. Lett. (277), 76e83. Huang, Q., Xi, G., Alamdar, A., et al., 2017. Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats. Environ Pollut. 229, 210e218. Huang, Q., Huang, H., 2011. Differential expression profile of membrane proteins in zebrafish (Danio rerio) brain exposed to methyl parathion. Proteomics 11, 3743e3756. Huang, Q., Huang, H., 2012a. Alterations of protein profile in zebrafish liver cells exposed to methyl parathion: a membrane proteomics approach. Chemosphere 87, 68e76. Huang, X., Huang, H., 2012b. Alteration of kidney membrane proteome of Mizuhopecten yessoensis induced low-level methyl parathion exposure. Aquat. Toxicol. 114e115, 189e199. Hulla, J.E., 2014. Metals. In: Hayes, A.W., Kruger, C.L. (Eds.), Hayes’ Principles and Methods of Toxicology. Taylor & Francis Group, Boca Raton, Florida, p. 849. Jongeneelen, F.J., 2014. A guidance value of 1-hydroxypyrene in urine in view of acceptable occupational exposure to polycyclic aromatic hydrocarbons. Toxicol. Lett. 231 (2), 239e248. Kang, S.W., Rhee, S.G., Chang, T.S., et al., 2005. 2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications. Trends Mol. Med. 11, 571e578. Kap-Soon, N., Do-Youn, L., Hak, C.J., et al., 2004. Protein biomarkers in the plasma of workers occupationally exposed to polycyclic aromatic hydrocarbons. Proteomics 4, 3505e3513.

Kim, B., Moon, P., Lee, J., et al., 2013a. Identification of potential serum biomarkers in mercury-treated mice using a glycoproteomic approach. Int. J. Toxicol. 32 (5), 368e375. Kim, J.H., Gil, H.W., Yang, J.O., et al., 2011. Serum uric acid level as a marker for mortality and acute kidney injury in patients with acute paraquat intoxication. Nephrol. Dial. Transplant. 26, 1846e1852. Kim, K., Jahan, S.A., Kabir, E., et al., 2013b. A review of airborn polycyclic aromatic hydrocarbons (PAHs) and their human health effects. Environ. Int. 60, 71e80. Kim, S.Y., Lee, H.M., Kim, K.S., et al., 2015. Non-invasive biomarker candidates for cadmium-induced nephrotoxicity by 2DE/ MALDI-TOF-MS and SILAC/LC-MS proteomic analyses. Toxicol. Sci. 148 (1), 167e182. Kim, Y., Jung, H., Gil, H., et al., 2012. Proteomic analysis of changes in protein expression in serum from animals exposed to paraquat. Int. J. Mol. Med. 30, 1521e1527. Kim, Y., Jung, H., Zerin, T., Song, H., 2013c. Protein profiling of paraquat-exposed rat lungs following treatment with Acai (Euterpe oleracea Mart.) berry extract. Mol. Med. Rep. 7, 881e886. Landrigan, P.J., Belpoggi, F., 2018. The need for independent research on the health effects of glyphosate-based herbicides. Environ. Health 17, 51. Levi, L., Lobo, G., Doud, M.K., et al., 2013. Genetic ablation of the fatty acid-binding protein FABP5 suppresses HER2-induced mammary tumorigenesis. Cancer Res. 73, 4770e4780. Min, L., He, S., Chen, Q., et al., 2011. Comparative proteomic analysis of cellular response of human airway epithelial cells (A549) to benzo(a) pyrene. Toxicol. Mech. Methods 21 (5), 374e382. Ogawa, R., Ishiguro, H., Kuwabara, Y., et al., 2008. Identification of candidate genes involved in the radiosensitivity of esophageal cancer cells by microarray analysis. Dis. Esophagus 21, 288e297. Pari, L., Murugavel, P., Sitasawad, S.L., et al., 2007. Cytoprotective and antioxidant role of diallyltetrasulfide on cadmium induced renal injury: an in vivo and in vitro study. Life Sci. 80, 650e658. Phark, S., Park, S., Chang, Y., et al., 2016. Evaluation of toxicological biomarkers in secreted proteins of HepG2 cells exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and their expression in the plasma of rats and incineration workers. Biochim. Biophys. Acta 584e593. Rocha, T.L., Rezende dos Santos, A.P., Yamada, A.T., et al., 2015. Proteomic and histopathological response in the gill of Poecilia reticulate exposed to glyphosate-based pesticide. Envrion. Toxicol. Pharmacol. 40, 175e186. Rodgman, A., Smith, C.J., Perfetti, T.A., 2000. The composition of cigarette smoke: a retrospective with emphasis on polycyclic components. Hum. Exp. Toxicol. 19, 573e595. Samuel, M., Sastry, K.V., 1989. In vivo effect of monocrotophos on the carbohydrate metabolism of freshwater shake head fish (Channa punctatus). Pestic. Biochem. Physiol. 34, 1e8. Sany, S.B.T., Hashim, R., Salleh, A., et al., 2015. Dioxin risk assessment: mechanisms of action and possible toxicity in human health. Environ. Sci. Pollut. Res. 22, 19434e19450. Satarug, S., Moore, M.R., 2004. Adverse health effects of chronic exposure to low-level cadmium in foodstuffs and cigarette smoke. Environ. Health Perspect. 112 (10), 1099e1103. Shin, Y., Kim, K., Kim, E., et al., 2017. Identification of aldo-keto reductase (AKR7A1) and glutathione-S-transferase (GSTP1) as novel renal damage biomarkers following exposure to mercury. Hum. Exp. Toxicol. https://doi.org/10.1177/0960327117751234. Sun, S., Wang, H., Zhao, G., et al., 2011. Complement inhibition alleviates paraquat-induced acute lung injury. Am. J. Respir. Cell Mol. Biol. 45, 834e842.

X. APPLICATIONS OF BIOMARKERS IN TOXICOLOGY

REFERENCES

Thompson, L.J., 2018. Lead. In: Gupta, R.C. (Ed.), Veterinary Toxicology; Basic and Clinical Principles, third ed. Academic Press/ Elsevier, Amsterdam, pp. 439e443. Tokar, E.J., Boyd, W.A., Freedman, J.H., et al., 2013. Toxic effects of metals. In: Klaasen, C.D. (Ed.), Casarett & Doull’s Toxicology, the Basic Science of Poisons, eighth ed. McGraw-Hill Education, China, pp. 986e999. Verma, N., Pink, M., Rettenmeier, A.W., et al., 2013. Benzo(a)pyrenemediated toxicity in primary pig bladder epithelial cells: a proteomic approach. J. Proteom. 85, 53e64. Wang, M., Wang, Y., Zhang, L., et al., 2013. Quantitative proteomic analysis reveals the mode of action for chronic mercury

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hepatotoxicity to marine medaka Oryzias melastigma. Aquat. Toxiocol. 130e131, 123e131. Wang, M.H., Wang, Y.Y., Wang, J., et al., 2011. Proteome profiles in medaka (Oryzias melastigma) liver and brain experimentally exposed to acute inorganic mercury. Aquat. Toxicol. 103 (3e4), 129e139. Wang, Y., Wang, D., Lin, L., et al., 2015. Quantitative proteomic analysis reveals proteins involved in the neurotoxicity of marine medaka Oryzias melastigma chronically exposed to inorganic mercury. Chemosphere 119, 1126e1133. Yeo, C.D., Kim, J.W., Kim, Y.O., et al., 2011. The role of pentraxin-3 as a prognostic biomarker in paraquat poisoning. Toxicol. Lett. 212, 157e160.

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