Detection of Carbofuran-Protein Adducts in Serum ... - ACS Publications

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Detection of Carbofuran-Protein Adducts in Serum of Occupationally Exposed Pesticide Factory Workers in Pakistan Tanzila Rehman,†,§,⊥ Mohd M. Khan,§,⊥,□ Muhammad A. Shad,‡ Mazhar Hussain,† Benjamin L. Oyler,§,■ Young Ah Goo,§,¶ and David R. Goodlett*,§ †

Institute of Chemical Sciences, ‡Department of Biochemistry, Bahauddin Zakariya University, Multan 60800, Pakistan Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201, United States

§

S Supporting Information *

ABSTRACT: This study was conducted to investigate the protein adducts with pesticides in a cohort of 172 factory workers that were exposed to a mixture of pesticides. The 35 samples showing considerable variation in biochemical parameters, i.e., butyrylcholinestrase (BChE), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), gamma-glutamyl transferase (GGT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP/ALKP), lactate dehydrogenase (LDH), creatine phosphokinase (CPK) enzymes, and controls were analyzed by reversed-phase nanoscale liquid chromatography tandem mass spectrometry (nLC−MS/MS) on an Orbitrap mass spectrometer employing a shotgun proteomics approach. Only protein adducts with carbofuran were found on serum proteins of these workers. These adducts were of carbofuran labeled lysine (Lys-142, Lys-183, Lys-287, and Lys-467), arginine (Arg-210, Arg-242, and Arg-256) from serum albumin, and serine (Ser-07, Ser-54, and Ser-150) from immunoglobulin proteins. The arginine residues (Arg-210, Arg-242, Arg-246, and Arg-434) from albumin were also found to be glycated in serum of workers showing a high level of glucose who also had glycated arginine (Arg-1120) modified with carbofuran in their tankyrase-1-binding protein. The number of tandem mass spectra of modified peptides increased with increasing time of exposure. This is the first report to demonstrate the presence of carbofuran-labeled albumin, immunoglobulin, and glycated arginine, which shows that lysine and arginine of human albumin and serine of immunoglobulin are covalently modified in the serum of workers that were occupationally exposed to carbofuran, and the modification is detectable by tandem mass spectrometry. These peptides modified with carbofuran can potentially be used as a biomarker of carbofuran exposure.

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INTRODUCTION Carbamate compounds are broad-spectrum pesticides that are commonly used to control insects, rootworms, nematodes, and beetles in agricultural settings across the world.1 One of the most commonly used carbamate compounds, carbofuran (2,3dihydro-2,2-dimethyl-7-benzofuranol N-methylcarbamate; Scheme 1), is responsible for the highest acute toxicity to humans as compared to any other insecticides used in agricultural settings.2 In animals and humans, carbofuran toxicity, like other carbamates, is characterized by reversible inactivation of acetylcholinesterase (AChE) that results in cholinergic symptoms.3 In addition to neurotoxic effects, carbofuran has also been reported to affect steroid metabolism in mammals.4 Recent epidemiological studies indicate that individuals with high levels of exposure to carbofuran and other carbamate pesticides may have increased risk for lung cancer and non-Hodgkins B-cell lymphoma.5,6 Acute intoxication symptoms, whether it is from gastrointestinal (GI) ingestion or inhalational exposure, include bronchorrhea, bronchospasm, bradycardia, diarrhea, emesis, lacrimation, miosis, salivation, urination, and some nicotinic symptoms such as muscle fasciculations and weakness.7,8 Also, patients with clinical cholinergic poisoning by carbamate exhibit oversweating, © 2016 American Chemical Society

labored breathing with symptoms of bronchorrhea and wheeze, and constricted pupils.9 Carbamates and organophosphous pesticides are highly effective inhibitors of butyrylcholinesterase (BChE) as they bind to the active site of serine, thereby blocking BChE’s enzymatic activities.10 Most recently, investigations on covalent organophosphates (OP) adduction of non-AChE protein targets in mice,11 guinea pigs,12 and humans13−15 have shown that circulating serum proteins may serve as new biomarkers of pesticide toxicants and nerve agent exposure. Because albumin is the most abundant protein in plasma (40 000 μg/mL), it is susceptible to xenobiotic binding and displays suitable traits for a biomarker even at a low level of exposure.16,17 Low plasma levels of albumin are associated with an increased rate of plasma protein glycation in diabetic patients.16,18 However, the rates of reactions of OP with albumin are slow compared to those with BChE; the higher concentration of albumin compensates for the slow reactivity and results in OP labeling in vivo.10,15,19 As shown in these studies, either the parent compounds or their metabolites that are capable of forming covalent bonds with Received: June 25, 2016 Published: September 22, 2016 1720

DOI: 10.1021/acs.chemrestox.6b00222 Chem. Res. Toxicol. 2016, 29, 1720−1728

Article

Chemical Research in Toxicology Scheme 1. Carbofuran Metabolism by CYP Enzymesa

a

The carbamate group of carbofuran (highlighted in red) forms covalent adducts with serine, lysine, or arginine residues, thereby adding a mass of 57.05 amu to the protein and releasing the hydroxybenzofuran ring structure in the process. signed consent forms, agreeing to participate in the study. These factories were formulating the pesticides mentioned in Table S1 of the Supplemental Information. Participants were from a bigger group of 180 occupationally exposed male workers who have been continuously working in pesticide formulating factories from 2 to 10 years and 50 control individuals with the same socioeconomic status but without previous occupational exposure to pesticides or any other industrial chemicals. The exposed workers were randomly selected based on their full time active involvement in preparation, packaging, and storage of pesticides in the pesticide formulating factories. The information regarding the work experience of the workers was taken from the factory records. All subjects were categorized into six groups based upon their timeline of exposure (G0−G5) from control to exposed individuals. The seven members that showed considerable variation in their biochemical parameters (details in next section) from each exposed group and control group were selected for this study. Sample Collection and Processing. Blood samples were collected by venipuncture procedure in serum separator tubes (gel barrier tubes; Becton Dickinson (BD) Franklin Lakes, NJ, United States) and were centrifuged at 1500 rcf at room temperature to isolate the serum (supernatant). The samples were further subjected to screening for hepatitis B and C using Hepatitis Rapid Kits (OraSure Technologies, Bethlehem, PA, United States). The eight subjects showing hepatitis B or C positive tests were excluded from the study. The concentrations of biochemical enzymatic parameters, including butyrylcholinestrase (BChE), serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), gammaglutamyl transferase (GGT), serum glutamic pyruvic transaminase (SGPT), alkaline phosphatase (ALP/ALKP), lactate dehydrogenase (LDH), and creatine phosphokinase (CPK), were estimated on a semiautomated Clinical Chemistry Analyzer, Microlab 300 (ELITech Group, Puteaux, France). The BChE level in serum was estimated using a commercially available kit (Randox Laboratories Ltd., Antrim, N. Ireland). The rest of the parameters were measured following the procedures provided for HUMAN kits (Human Diagnostics Worldwide, Germany). Further, total protein estimation was accomplished using a bicinchoninic acid (BCA) assay on a BioTek Eon plate reader (BioTek Instruments Inc., Winooski, VT, United States), essentially following the manufacturer’s instructions (Thermo Fisher Scientific Inc., Rockford, IL, United States). For the present study, a total of 42 participants (seven members from each exposure group of 2, 4, 6, 8, and 10 years) showing considerable variation in the aforementioned biochemical parameters and control group were selected for further serum analysis by reversed-phase nanoscale LC−MS/MS. All samples were properly refrigerated and stored at −80 °C to avoid any sample degradation prior to LC−MS/MS; processed serum samples were thawed only prior to TCA precipitation and trypsin digestion. Trichloroacetic Acid (TCA) Precipitation of Samples. Following the BCA analysis of the samples, 100 μg of protein per sample was used for trypsin digestion without further sample treatment or depletion. Briefly, serum proteins were precipitated by mixing sample and TCA at a 50:50 ratio using ice-cold, freshly made 20% (w/v) TCA solution.34 After brief vortex for 15 s, sample tubes were left on ice for further precipitation (∼30 min). Samples were then centrifuged to pellet precipitated proteins at 14 000 rcf for 15 min at 4 °C. After the

proteins modify the target proteins. Due to the adverse effects of carbofuran on human health and domestic and wild animal populations, it was banned in the United States but is still being used in many countries of the world.20,21 Agriculture is the largest sector of Pakistan’s economy and contributes over 23% to its Gross Domestic Product (GDP), employing 42% of the total labor force. The industrial state of Multan is the hub of pesticide formulation units where, according to a prefeasibility study for the pesticide industry report published in 2006, a large number of workers are occupationally exposed to different pesticides as occupational exposure is the most common cause of pesticide poisoning.22 In developing countries, occupational illness is common because its prevention is deemed impractical and expensive and there are no regulations in place to require use of safety equipment to minimize or prevent exposure.23,24 Safety instructions provided for the pesticides are often written in unfamiliar languages to workers; as many farmers are illiterate, safe handling protocols without proper guidance and clarifications are difficult to follow.25 For instance, after coming into contact with pesticides, it is difficult to “wash off at once” when there is no water available. Because of the pesticide-associated potential health challenges, the World Health Organization (WHO) classifies their poisonings as class I (extremely toxic) to class III (slightly hazardous).25 Use of liquid chromatography with tandem mass spectrometry (LC−MS/MS) is a valuable method for disease diagnosis and research, including discovery-based proteomic investigations,26 metabolomic studies,27 drug discovery,28 and biomarker screening and validation.29 LC−MS/MS-based proteomics does not require an individual-based baseline activity measurement, thereby eliminating the need for pre-exposure bleeds and can be easily employed to detect protein modifications with a very high throughput. Previously, some in vitro and ex vivo studies demonstrated covalent binding of OPs to albumin14,30,31 and BChE,32,33 but there is a paucity of information in the literature regarding the binding of carbamates with noncholinesterase proteins. To our knowledge, no studies are available reporting carbofuran-serum protein labeling in occupationally exposed workers. Here, we report detection of covalent adducts of carbofuran with the major serum proteins albumin and immunoglobulin in sera collected from workers occupationally exposed to pesticides using an LC−MS/MS shotgun proteomic approach.

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METHODS

Study Design. The study was approved by the Board of Advanced Studies and Research (BASR), Bahauddin Zakariya University, Multan, Punjab, Pakistan. The participants were recruited from the pesticide formulating factories situated in Industrial Sate Multan (Pakistan) and 1721


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Chemical Research in Toxicology

Data Interpretation, Visualization, and Plotting. Statistical significance for the analysis of biochemical parameters between the control and exposed groups was subjected to analysis of variance (ANOVA) together with Tukey’s multiple comparison test (p ≤ 0.05) using GraphPad Prism 5. Tandem mass spectral data features, as determined by TPP for the modified peptides, were exported to an open-source program, QtiPlot (www.qtiplot.com), and further rendered and annotated in Inkscape (www.inkscape.org). A relationship between exposure and the degree to which carbofuran-protein adducts were observed was produced by calculating the percentage modification of a peptide relative to the same unmodified peptide sequence (i.e., the percentage between the tandem mass spectra that were modified for a given peptide sequence and the total number of tandem mass spectra for that respective peptide versus the number of years of exposure). Bar graphs were drawn showing modification probability (%) vs exposure using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, United States).

supernatant was discarded, the pellet was washed twice with cold acetone (100 μL) and centrifuged again. The air-dried pellet (∼15 s) was resuspended in 250 μL of reduction/denaturation buffer composed of 7.5 mM Tris(2-carboxyethyl)-phosphine hydrochloride (TCEP), 8 M urea, and 100 mM ammonium bicarbonate pH 8.0 (ambic) and further processed using filter-aided sample preparation (FASP).34 Protein Digestion, Alkylation, and Desalting using the FASP Method. The FASP method for proteomics sample preparation was adapted from Wiśniewski et al.34 Briefly, resuspended protein samples in urea-ambic-TCEP solution were loaded into an Amicon Ultra-0.5 mL 3K MWCO filter (EMD Millipore, Darmstadt, Germany), followed by dry-bath incubation at 37 °C for 1 h. Reduced, denatured samples were centrifuged at 14 000 rcf for 15 min at room temperature. The protein solution, remaining in the filter, was again dissolved in 250 μL of 8 M urea, 100 mM ambic, and 50 mM iodoacetamide (IAM) and left in the dark for 1 h at room temperature. Alkylated samples were centrifuged at 14 000 rcf for 15 min, and 25 μL of 0.5 M DL-dithiothreitol (DTT) was added to quench residual IAM. Further centrifugation at 14 000 rcf for 15 min was achieved. Samples were further washed four times with 300 μL of 50 mM ambic to remove salts and urea. The concentrate was then collected in an Eppendorf tube and subjected to a proteolytic digestion by adding trypsin (Promega, mass spectrometry grade, Madison, WI, United States) in 50 proteins to 1 trypsin weight ratio and leaving the sample for an overnight incubation in a dry bath at 37 °C. The peptide sample was acidified (pH < 4) to deactivate trypsin using a final concentration of 1% trifluoroacetic acid. The acidified tryptic samples were dried completely with a Speedvac (Thermo Scientific, Waltham, MA, United States) and resuspended in 95.0% water/5.0% acetonitrile/0.1% formic acid to a final concentration of 1.0 μg/μL protein. Samples were either immediately analyzed or stored at −80 °C until LC−MS/MS data acquisition. LC−MS/MS Data Acquisition and Processing. After digestion, peptides were analyzed by LC−MS/MS with an Alliance 2695 pump (Waters Corporation Milford, MA, United States), a PAL HTS autosampler (Leap Technologies, Carrboro, NC, United States), and an LTQ Orbitrap mass spectrometer (Thermo Fisher, San Jose, CA, United States). Peptide separation was achieved on an in-house fabricated laser pulled tip column consisting of a 100 μm ID × 150 mm length of fused silica (Polymicro Technologies, Phoenix, AZ, United States) packed with YMC Triart 120 Å pore size, C18, 1.9 μm particles (YMC America, Inc., Allentown, PA, United States). An estimated 2 μg of peptide mixture was injected per LC−MS/MS run at a 0.45 μL/min flow rate using an initial eluent of 0.1% formic acid in water. Peptides were eluted with a 60 min linear gradient from 3 to 30% mobile phase B consisting of 0.1% formic acid in acetonitrile. Precursor ion scans from 400−1200 m/z were obtained at 60 000 resolution (at 400 m/z). Collision-induced dissociation (CID) spectra were acquired on the 10 most abundant ions for each scan in data dependent acquisition mode. Dynamic exclusion was set for 30 s, and CID spectra were obtained only for ions with a charge state greater than 1. The precursor ions were fragmented using 35% relative collision energy with helium as the collision gas in the linear ion trap. Individual raw data files were converted to mzML format with msconvert35 and processed with the Trans-Proteomic Pipeline version 4.8.36 Comet was used to search the Swissprot Homo sapiens database that was concatenated with a set of common contaminants. Variable modifications consisting of oxidation of methionine and a fixed modification of carbamidomethylation of cysteine were allowed. Variable modification for the analysis of adducts of pesticides with proteins (Table S1) was added to the search. The number of modifications per peptide was given as 3. Enzyme specificity was set to trypsin, allowing for two missed cleavage sites per peptide. MS1 precursor ion mass tolerance was 50 ppm, and MS2 product ion mass tolerance was 0.4 Da. Peptide spectra matched (PSM) to theoretical spectra calculated from the database were validated using Peptide Prophet, and peptides were assembled into protein groups with Protein Prophet.

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RESULTS Carbofuran is a very toxic carbamate compound and among the most readily absorbed insecticides as compared to several wellknown pesticides including carbaryl, chlorpyrifos, dichlorodiphenyltrichloroethane (DDT), malathion, and parathion. Upon rapid absorption through the skin, lungs, GI tract, and mucous membranes, carbofuran is distributed to all organs and is rapidly eliminated.3,37,38 Over the years, carbofuran poisoning has been extensively studied by many researchers in humans, insects, mice, and plants. 39−42 In humans, carbofuran metabolism is mediated by the cytochrome P450 (CYP3A4) enzyme, which is one of the most abundant drug-metabolizing CYP isoforms in the human liver that metabolizes endogenous compounds. The main pathway of oxidative metabolism of carbofuran in animals, insects, and plants appears to consist of hydroxylation of a benzylic carbon to yield a major ring oxidation metabolite, 3-hydroxycarbofuran, and two minor metabolites (Scheme 1). The metabolites may form a proteincarbofuran adduct when reacting with the hydroxyl group of serine and the primary amine of lysine or arginine (Figures 1 and 2). Biochemical Analysis. Table 1 presents the levels of biochemical parameters determined in serum of the individuals of control and exposed groups. Descriptive quantitative data were expressed as mean ± SD, and Tukey’s multiple comparison test was performed to determine the statistically significant differences observed between the control subjects and subjects with varying years of exposure. Specifically, a significant decrease (P < 0.0001) was observed in butyrylcholinesterase levels of all exposed groups versus the control group (Table 1). Also, the ALKP level showed a significant increase (p < 0.001) in individuals of G1 to G3 and in G4 and G5 (p < 0.0001) from that of the control. Significantly higher levels of CPK and LDH were observed in members of G3 (p < 0.001), G4, and G5 (p < 0.0001), respectively. The increase in levels of these enzymes, we speculate, is a result of the stress of pesticide exposure. In liver function enzymes SGOT, SGPT, and GGT, a significant difference (p < 0.05 to p < 0.001) was observed in groups G3−G5, as shown in Table 1. Finally, a significant variation in the total proteins of exposed individuals (p < 0.05) was observed in members of highest exposure group (G5). We speculate that the chronic pesticide exposure leads to changes in kidney and liver function enzymes as well as cholinesterases in occupationally exposed workers. Identifying Carbofuran Adducted Sites on Human Serum Albumin (HSA) and Immunoglobulin. The fact that 1722


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Figure 2. Carbofuran modification prevalence. The percentage of modifications at a given amino acid (*) is plotted as a function of exposure (in years) for (A) lysine, (B) serine, and (C) arginine.

Figure 1. Tandem mass spectra of carbofuran-modified tryptic peptides from serum of occupationally exposed workers. (A) In HSA, Lys-142 is modified with carbofuran in AEFAEVSKLVTDLTK. (B) In immunoglobulin proteins, Ser-150 is modified with carbofuran in TPEVCVVDVSHEDPEVK. (C) In HSA the arginine residue for the peptide with sequence RHPDYSVVLLLR is modified at the first arginine. Amino acid(s) detected as modified are shown in bold and underline format.

observed carbofuran modification sites at lysine and arginine residues in HSA and serine residues in IgG (Table 2). The selected tandem mass spectra showing modification at Lys, Arg, and Ser are shown in Figure 1, and the rest of the modified peptides are presented in the Supporting Information, Figures S1−11. The prevalence of carbofuran-protein adduct formation was assessed by spectral counting, which is a common method for relative quantification from DDA-generated proteomic data, of the modified peptides in each individual (Figure 2). Figure 1 (A-C) depicts the three peptides AEFAEVSKLVTDLTK, TPEVTCVVVDVSHEDPEVK, and RHPDYSVVLLLR showing modification by carbofuran at lysine, serine, and arginine residues, respectively. Peptide AEFAEVSKLVTDLTK in Figure 1A shows modification at Lys 142. The singly charged ions in panel A show y8 and b8 ion masses of 974 and 919 m/z, respectively. These are the masses of the ion plus 57 Da for the mass of carbofuran. An extensive b ion (b8−b14) and y ion (y8−y11) series indicates that most of the b and y ions are supporting the presence of a modification at Lys 142. In total, four peptides (Table 21−4) modified by carbofuran at lysine residues have been found in all five groups of exposure. The percentage of modified peptides with the sequences YICENQDSISSKLK and AVMDDFAAFVEKCCK ranged from 57 to 100%, while for the peptides AEFAEVSKLVTDLTK and VFDEFKPLVEEPQNLIK, the range was from 28 to 100% across groups of workers exposed from 2 to 10 years. A direct correlation was observed between the

organophosphorus and carbamate pesticides potentially could modify serine hydrolases provides a means for identifying the specific pesticide binding to noncholinesterase proteins causing observable post-translational protein modifications.43 Albumin has been reported to bind with OP even with low dose administration to mice and in humans.19,44,45 Many studies have shown that pesticides could cause impairment or suppression of the immune and respiratory systems by targeting the function of cellular, subcellular, or molecular components.46,47 LC-purified and separated peptides from trypsin digest all serum proteins from occupationally exposed workers, who were routinely exposed to carbofuran in pesticide formulating factories, were analyzed by tandem MS in an Orbitrap mass spectrometer. To confirm the expected labeling sites for carbofuran, tandem mass spectra generated by datadependent acquisition (DDA) during the LC separation were searched against a protein sequence database using the Comet search engine of the trans-proteomic pipeline (TPP).36 Those tandem mass spectra suspected of originating from carbofuran adducted peptides were manually validated to confirm or refute the suggested amino acid modifications. From these results, we 1723


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Chemical Research in Toxicology Table 1. Effect of Pesticides on Biochemical Parameters of Occupationally Exposed Workersa parameter G0 G1 G2 G3 G4 G5

(2Y) (4Y) (6Y) (8Y) (10Y)

BChE (U/L) 26700 ± 3390 16100 ± 3170*** 11000 ± 6620*** 7200 ± 3950*** 4060 ± 2430*** 3860 ± 1830***

ALKP (U/L) 101 188 189 196 259 270

± ± ± ± ± ±

22.4 62.8** 26.5** 49.5** 29.8*** 30.6***

CPK (μg/L) 46.6 ± 8.80 65.0 ± 15.9 67.4 ± 22.1 107 ± 32.6** 132 ± 26.7*** 134 ± 49.4***

LDH (U/L) 149 221 242 296 314 314

± ± ± ± ± ±

SGOT (U/L)

38.5 40.4 61.7 53.5** 113*** 56.7***

22.0 38.3 39.0 47.4 54.2 58.5

± ± ± ± ± ±

7.40 20.6 14.7 13.1** 10.1** 8.40**

SGPT (U/L) 21.8 31.1 27.1 45.8 50.2 52.5

± ± ± ± ± ±

5.80 14.0 17.8 18.7* 18.8** 13.9**

g-GT (μ/L) 11.2 31.4 32.8 44.7 48.4 53.7

± ± ± ± ± ±

6.70 6.50 15.8 31.8* 7.90** 12.7**

protein (μg/μL) 111 ± 10.9 107 ± 5.80 107 ± 29.4 105 ± 10.3 98.8 ± 11.6 85.1 ± 20.4*

a Values are mean ± SD; *P < 0.05, **P < 0.001, and ***P < 0.0001 indicate values significantly different from control (G0) and exposed (G1−G5) groups applying Tukey’s multiple comparison test (U: unit).

Table 2. Identification of Carbofuran Pesticide Adduct Sites in Serum of Occupationally Exposed Workersa serial

protein name

1

tr|B7WNR0|B7WNR0_HUMAN serum albumin tr|B7WNR0|B7WNR0_HUMAN serum albumin tr|B7WNR0|B7WNR0_HUMAN serum albumin tr|B7WNR0|B7WNR0_HUMAN serum albumin sp|P01775|HV314_HUMAN Ig heavy chain V−III region sp|P01766|HV305_HUMAN Ig heavy chain V−III region sp|P01620|KV302_HUMAN Ig kappa chain V−III region sp|P01857|IGHG1_HUMAN Ig gamma-1 chain C region > tr|B7WNR0|B7WNR0_HUMAN serum albumin > tr|B7WNR0|B7WNR0_HUMAN serum albumin > tr|B7WNR0|B7WNR0_HUMAN serum albumin tr|B7WNR0|B7WNR0_HUMAN serum albumin tr|B7WNR0|B7WNR0_HUMAN serum albumin tr|B7WNR0|B7WNR0_HUMAN serum albumin tr|B7WNR0|B7WNR0_HUMAN serum albumin > sp|Q9C0C2−2|TB182_HUMAN isoform 2 of 182 kDa tankyrase-1binding protein

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 a

peptide position

peptide sequence

position of the modified residue

modification site

MH+

m/z

score

YICENQDSISSKLK

172−185

183

Lys + Carb

1741.8426

871.425

0.84

VFDEFKPLVEEPQNLIK

282−298

287

Lys + Carb

2102.1169

1051.562

1.00

AEFAEVSKLVTDLTK

135−149

142

Lys + Carb

1707.9164

854.462

0.82

AVMDDFAAFVEKCCK

455−469

467

Lys + Carb

1847.8125

924.410

0.70

AVQLLESGGGLVQPGGSLR

1−19

07

Ser + Carb

1895.0345

948.021

1.00

EVQLVESGGGLVQPGGSLR

1−19

07

Ser + Carb

1939.0244

970.016

0.99

47−55

54

Ser + Carb

1036.5786

518.793

0.70

139−157

150

Ser + Carb

2196.0489

1098.529

0.96

RHPYFYAPELLFFAK

54−68

54

Arg + Carb

1956.0167

652.677

1.00

RHPYFYAPELLFFAKR

54−69

69

Arg + Carb

2112.1178

704.711

0.90

RHPDYSVVLLLR

361−372

361

Arg + Carb

1524.865

508.959

1.00

AWAVARLSQR

237−246

242

Arg + MGO

1211.6643

606.336

0.78

LSQRFPK

243−249

246

Arg + MGO

929.5203

465.264

0.78

LDELRDEGK

206−214

210

Arg + MGO

1128.5531

564.780

0.98

FQNALLVR

427−434

434

Arg + MGO

1014.5730

507.790

1.00

DFCIEASER

1112−1120

1120

Arg + MGO + Carb

1237.5108

619.259

0.90

LLIYGASSR TPEVTCVVVDVSHEDPEVK

Where Carb = carbofuran and MGO = Methylglyoxal. Score signifies the probability that the modified peptide sequence is correctly assigned.

dehydrated serine was observed. Our findings are in agreement with those of Li et al.,51 who also did not find ions for dehydrated serine in their study of carbofuran poisoning by analysis of BChE. From spectral counting analysis, the four peptides TPEVTCVVVDVSHEDPEVK, LLIYGASSR, AVQLLESGGGLVQPGGSLR, and EVQLVESGGGLVQPGGSLR belonging to Ig gamma-1, Ig kappa, and Ig heavy chains respectively, have been observed in all exposed groups. Prevalence of modification (Figure 2B) of the two peptides TPEVTCVVVDVSHEDPEVK and LLIYGASSR at serine residues was from 14 to 28% for groups with 2−4 years of exposure. The prevalence of modification of the peptide AVQLLESGGGLVQPGGSLR at serine residues was found to be 14 to 71% in groups from 2 to 10 years of exposure. The modified peptide EVQLVESGGGLVQPGGSLR has been observed in individuals of only higher exposure groups.

percentage of a given peptide that was modified and increasing time of exposure (Figure 2). Chou et al. reported that the two peptides AEFAEVSK and YICENQDSISSK were modified by alkaloids,48 whereas Ding et al.49 reported the modification of carbamate (57 Da) at a serine residue in the same peptide, AEFAEVSK. Carbofuran labeling at serine residues (Table 25− 8) has been observed in four peptides of different chains of IgG proteins. The peptide TPEVTCVVVDVSHEDPEVK from the IGg-1 chain C region shows modification at Ser-150 (Figure 1B). The mass of singly charged y8 ion (997 m/z) is equal to the sum of y7 (853 m/z), serine (87 m/z), and the added mass of carbofuran residue (57 Da). A variety of singly charged y ion fragments support the labeling assignment. Prominent b ion fragments confirm the identity of the peptide. Unlike OPmodified serine,32,49,50 we observed that carbofuran-modified serine does not undergo β-elimination as no additional ion for 1724


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Chemical Research in Toxicology Scheme 2. Formation of Glycated Arginine and Its Reaction with Carbofuran Metabolite

However, Chou et al. also reported the modification of the peptide TPEVTCVVVDVSHEDPEVK with alkaloids at threonine and cysteine residues, but their study employed an in vitro system.48 Peptide RHPDYSVVLLLR in Figure 1C is modified with carbofuran at Arg-361 (albumin) of pesticide formulating workers. Masses for singly (b2+−b8+) and doubly charged b2+2 and series (b4+2−b7+2 and b9+2−b11+2) ions indicate the presence of carbofuran modification at the first arginine residue. An extensive singly charged y ion series (y1+ to y8+) also supports modification of this peptide as do doubly charged ions (y5+2, y7+2, and y10+2). These peptides have been detected in sera of workers ranging from low exposure to higher exposure with a prevalence of modification ranging from 14 to 28% (Figure 2C). The modifications of Arg-54 and Arg-69 in peptide RHPYFYAPELLFFAKR (Figures S7 and S8) were found throughout the exposed groups from 2 to 10 years of exposure. The peptide RHPDYSVVLLLR was observed in the serum samples of only higher exposure groups (G3−G5). In fact, Ding et al. in 200849 reported the modification of Tyr-148 by OP at the peptide RHPYFYAPELLFFAKR. In light of the earlier work done on iso(thio)cyanates-protein adduct kinetics and evaluation of nucleophilicity of the amino acid moieties, we detected carbofuran adducts on Lys and Arg as well, which are not the best nucleophiles present in proteins but are leading the most stable adducts in addition to adducts on serine.52,53 This is in conformity with the work of Karlsson et al.,53 who showed that lysine adducts were stable for period of time longer than, for instance, cysteine adducts. Identifying Glycated Arginine and its Carbofuran Adduct Sites in Diabetic Workers. Reactive carbonyl groups of a reducing sugar react with neutrophilic free amino groups of proteins to form a reversible Schiff base. Through rearrangement, these Schiff bases lead to the formation of advanced glycation end products (AGEs).54,55 Methylglyoxal, a potent arginine-directed glycating agent, is an important precursor of AGEs in physiological systems, which reacts with albumin to form mainly the arginine-derived hydroimidazolone AGE, N5(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), argpyrimidine, the lysine-derived AGEs, N(epsilon)(carboxymethyl)lysine (CEL), and methylglyoxal-derived lysine dimer (MOLD).54 In HSA, researchers have identified five arginine residues (i.e., Arg-114, Arg-186, Arg-218, Arg-410, and Arg-428) as susceptible to modification by methylglyoxal (Scheme 2).51

In our study, in sera of occupationally exposed workers that were suffering from hyperglycaemia, four modification sites modified with MG-H1 were also identified by tandem mass spectrometric analyses (Table 2). Additionally, Arg-242 of HSA was found to be glycated in the peptide AWAVARLSQR (Figure 3A). The major peak of y6+ at 784 m/z indicates the

Figure 3. Tandem mass spectra of carbofuran-modified tryptic peptides in serum of occupationally exposed workers who also showed increased blood glucose levels. Shown are the modifications for (A) Arg-242 of HSA glycated at AWAVARLSQR with methylglyoxyl, increasing the peptide mass by 54 Da, and (B) carbofuran modification of glycated arginine, leading to an increase of 111 Da on peptide DFCIEASER of tankyrase-1-binding proteins.

existence of glycated arginine. A y ion series (y5+−y8+) and a b ion series (b6+−b9+) support the identification of this peptide as modified. Additional support for the presence of glycated arginine in the peptide comes from characteristic masses at 713 and 709 m/z. The arginine residues Arg-246, Arg-210, and Arg434 from the peptides of HSA (i.e., LSQRFPK, LDELRDEGK, and FQNALLVR, respectively), have also been found in a glycated state (Figures S9−S11) in diabetic workers. The advanced glycation products are susceptible to react further 1725


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Chemical Research in Toxicology with proteins55 or with locating agents like 6-aminoquinolyl-Nhydroxysuccinimidyl-carbamate (AQC).54 Also, low plasma levels of albumin are correlated with an increased rate of glycation of plasma proteins in diabetic patients.18 Advanced glycation end products (AGEs) are formed as a part of aging; AGE modification is further accelerated in diabetes.56 Recently, MS-based proteomic analysis of circulating immune complexes from diabetic mice showed elevated levels of serum albumin in plasma, further the circulating immune complexes were found to be AGEmodified.57 Long-term exposure of carbofuran to the diabetic workers also showed the carbofuran adducts formation on glycated arginine, as suggested in Scheme 2. Finally, a tandem mass spectrum from the peptide DFCIEASER showed a modification (mass shift of 111 Da) at Arg-1120 of tankyrase-1binding protein. Labeling of carbofuran on arginine is supported by the y ion series (y1+−y5+). The identity of the peptide is also supported by the b4−b8 ions (Figure 3B).

key information about the health of the subject, the extent of exposure, and possibly the origin of the exposure. Alternatively, we propose that one could monitor occupationally exposed workers using a discovery-type process as done here using LC− MS/MS without any hypotheses about what to expect. This would have the advantage of allowing new discoveries to take place while also monitoring exposure via adduction of highly abundant proteins HSA and IgG, which are easily detected, as shown here.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.6b00222. Table S1 shows the list of residue masses of the amino acids modified, mass shifts by pesticides searched, and the total mass of adducts used for modification search, and Figures S1−S11 show the mass spectra for carbofuran and MGO labeling as found at arginine, lysine, and serine residues (PDF)

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DISCUSSION Human serum can serve as an ideal source for biomarkers of exposure due to the ease of sample collection and the wide range of proteins held within the serum compartment. In terms of monitoring occupational exposure of pesticides, albumin is a good target because it readily forms adducts of xenobiotic compounds such as pesticides even at low pesticide exposure.44 In addition, serum proteins are relatively long-lived in the body and can be detected for much longer periods of time compared to highly reactive pesticide parent compounds and metabolites. Our study shows carbofuran labeling of 11 peptides belonging to HSA and Ig with prominent modifications at lysine, serine, and arginine residues. Spectral counting of modified peptides for lysine, arginine, and serine peptides showed that the number of modified peptides in sera of pesticide exposed workers increased with increased time of pesticide-handling occupation. Because protein turnover occurs in weeks, it is unclear why workers employed for longer periods of time, with longer total exposure times, would display higher levels of serum protein adduction. It is possible that the cumulative effect of exposure on a daily basis allows more adducted protein to remain in the system. It is also possible that chronic exposure to pesticides affects protein degradation rates in vivo. Finally, it is indeed possible that the protein adducts themselves limit the degree to which polypeptides can be degraded and recycled. However, all of these possibilities are completely conjecture. We did not investigate the underlying processes, which caused this observation in this study. Additionally, we found four peptides glycated at arginine with methylglyoxal and the glycated arginine modified with carbofuran at peptide DFCIEASER in tankyrase-1-binding proteins in diabetic workers that showed high blood glucose levels. Together with albumin, other carbofuran-modified proteins may serve as potential biomarkers for pesticide exposure. Amino acid residues adducted with pesticides in proteins with no active-site serine suggest that pesticides also modify other nonmetabolizing proteins with potential to produce deleterious biological outcomes in chronically exposed workers. As the proteins we detected to be adducted were all from blood, they could be used as biomarkers of exposure by MS-based methods such as multiple reaction monitoring or immuno-affinity type assays. In fact, various blood proteins already serve as biomarkers of exposure, effect, or susceptibility.58 These circulating proteins can be analyzed to provide

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], Phone: +1-410-7061490. Present Addresses ¶

Y.A.G.: Proteomics Center of Excellence, Northwestern University, Chicago, Illinois 60611, United States. □ M.M.K.: School of Medicine, University of Maryland, Baltimore, Maryland 21201, United States. ■ B.L.O.: Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, Baltimore, Maryland 21202. Author Contributions ⊥

T.R. and M.M.K. contributed equally to this work.

Funding

T.R. is thankful to the Higher Education Commission (HEC) of Pakistan for financial support. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We strongly appreciate the technical support of Brian Hampton, School of Medicine, UMB, and valuable suggestions from the members of the Goodlett Laboratory.

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ABBREVIATIONS LC−MS/MS, reverse phase liquid chromatography−tandem mass spectrometry; OP, organophosphates; CYP, Cytochrome P450

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REFERENCES

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