ToxSci Advance Access originally published online on May 21, 2007
Toxicological Sciences 2007 98(2):427-435; doi:10.1093/toxsci/kfm126
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Globin S-Propyl Cysteine and Urinary N-Acetyl-S-Propylcysteine as Internal Biomarkers of 1-Bromopropane Exposure
* Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2561
Shanghai Institute of Planned Parenthood Research, WHO Collaborating Center for Research Reproduction, Shanghai 200032, China
Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
1 To whom correspondence should be addressed at Department of Pathology, Vanderbilt University Medical Center, 1161 21st Ave S., Nashville, Tennessee 37232-2561. Fax: (615) 343-9825. E-mail: holly.valentine{at}vanderbilt.edu.
Received May 4, 2007; accepted May 8, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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1-Bromopropane (1-BP), an alternative to ozone-depleting solvents, is a neuro and reproductive toxicant in animals and humans. In this study, the dose responses for urinary AcPrCys and S-propylcysteine (PrCys) adducts on globin and neurofilaments were determined as a function of 1-BP exposure level and duration in the rat; and globin PrCys adducts and urinary AcPrCys were quantified in samples obtained from workers in a 1-BP production facility. Rats were exposed to 1-BP by inhalation for 2 weeks at 0, 50, 200, or 800 ppm and to 1-BP at 0 or 50 ppm for 4 weeks. After the 4-week exposures ended, half of the animals were euthanized immediately and half euthanized 8 days later. Urinary AcPrCys was measured using liquid chromatography–tandem mass spectrometry (LC/MS/MS) and gas chromatograph–mass spectrometry (GC/MS); and PrCys adducts were determined on globin and neurofilaments using LC/MS/MS. In rats, PrCys adduct and urinary AcPrCys levels demonstrated a linear dose response relative to exposure level. PrCys globin adducts demonstrated a linear cumulative dose response over the 4-week exposure period. Elimination of AcPrCys appeared biphasic with detectable levels still present in urine up to 8 days postexposure. A significant increase in globin PrCys adducts was observed in the 1-BP workers relative to control workers; and urinary AcPrCys increased with increasing 1-BP ambient exposure levels. The results of these studies demonstrate the ability of 1-BP to covalently modify proteins in vivo and support the potential of urinary AcPrCys and globin PrCys adducts to serve as biomarkers of 1-BP exposure in humans.
Key Words: 1-bromopropane; protein adduct; globin; neurofilaments; mercapturate; biomarkers.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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Along with tighter environmental regulations on production of ozone-depleting solvents such as chlorofluorocarbons or 1,1,1-trichloroethane, alternatives such as 1-BP (n-propyl bromide, 1-BP, CAS No. 106-94-5) have been introduced. 1-BP is currently used as a solvent vehicle for spray adhesives (Ichihara et al., 2002
) and cleaning and degreasing agent for metal components of precision instruments, metals, electronics, optical instruments, and ceramics (Ichihara, 2005). Human exposure, through production and use of 1-BP as a solvent, will likely increase unless less expensive, less toxic alternatives are developed. Recent animal studies (Ichihara et al., 2000a,b; Wang et al., 2002, 2003; Yamada et al., 2003) and reports of neurological and reproductive findings in exposed workers (Ichihara et al., 2002, 2004a,b; Sclar, 1999) have demonstrated the neurotoxicity and reproductive toxicity of 1-BP. Subchronic or chronic exposures may produce symptoms in workers that differ with those seen having acute exposures. Some of the symptoms observed in humans such as anxiety, irritability, headache, nausea, ataxia, or feeling drunk may be at least partially explained by Br– intoxication, but other symptoms such as muscle weakness, paresthesia, numbness, urinary incontinence, and memory disturbances have not been attributed to Bromism by previous studies (Hanes and Yates, 1938; Harney et al., 2003; Ichihara et al., 2004b, 2005; Majersik et al., 2007; Trump and Hochberg, 1976). A toxic mechanism other than Br– intoxication should be considered to fully explain peripheral and/or central nervous system injury caused by exposure to 1-BP. Occupational exposure monitoring in factories has been conducted in an effort to link exposure levels to adverse effects. Unfortunately, several problems occur in occupational exposure monitoring. In most occupational settings, it is unrealistic to monitor an individual's serial and long-term personal exposure levels. In addition, one time monitoring may not reflect long-term exposures if workers rotate tasks from high to low exposure areas during the course of a day or week.
Although 1-BP and its metabolites have had promising results as biomarkers for animal and human exposure (B'Hymer and Cheever, 2004, 2005; Garner et al., 2006), there is currently no consensus on the most appropriate biomarker or methodology for assessing human exposure to 1-BP. In addition, studies evaluating the dose response or elimination kinetics of biomarkers are lacking. Two studies investigating the dose response for production of N-acetyl-S-propylcysteine (AcPrCys) in rat urine and S-propylcysteine (PrCys) adduct formation on rat hemoglobin and neurofilament protein as a function of 1-BP exposure level and duration are presented in this paper. In the first study, rats were administered 1-BP by inhalation every day for 2 weeks at 0, 50, 200, or 800 ppm, 8 h/day. Urine was collected during the first full day of exposure and the level of urinary AcPrCys was measured by liquid chromatography–tandem mass spectrometry (LC/MS/MS) and gas chromatography–mass spectrometry (GC/MS). Following the final exposure, the PrCys adduct levels on globin and neurofilaments were determined using LC/MS/MS. In the second study, rats were exposed to 1-BP 50 ppm or filtered air over 4 weeks, 8 h/day, 5 days/week. Urine was collected before the first exposure each week and both blood and urine collected at the end of the fifth day of exposure each week. At the end of 4 weeks, urine was collected 48 h after the final exposure and then daily for 7 more days to gain a first approximation for the elimination rate of urinary AcPrCys after repeated exposure. Urinary ACPrCys and globin PrCys adduct levels were determined using LC/MS/MS and were correlated with duration of exposure up to 4 weeks. Elimination kinetics of the metabolite and protein adducts were also evaluated. In a third study, blood and urine samples were obtained from factory workers in a 1-BP production facility and the PrCys adducts on their globin determined by LC/MS/MS and their urinary AcPrCys levels determined by GC/MS and compared to ambient 1-BP levels determined by personal air monitoring devices.
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MATERIALS AND METHODS |
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Chemicals
1-BP used for animal inhalation exposures (99.81% purity) was a gift from Tosho Co., Japan. 1-BP-d7 was obtained from Isotec (Miamisburg, OH). N-acetylcysteine, ethyl acetate, cysteine, and diazald (precursor to diazomethane) were obtained from VWR Scientific (West Chester, PA). 4-t-Butyldimethylsiloxy-3-pentene-2-one, pyridinyl isothiocyanate (PyITC), triethylamine, and 1-BP used for chemical syntheses were obtained from Aldrich Chemical Co. (Milwaukee, WI). Deuterochloroform (CDCl3) was obtained from Sigma Chemical Co. (St Louis, MO).
Chemical Syntheses
AcPrCys and N-acetyl-S-(heptadeuteriopropyl)cysteine.
N-acetylcysteine (1.64 g, 10 mmol) was dissolved in 2N NaOH (10 ml) and stirred with 1-BP (1 ml, 11 mmol) in ethanol (3 ml) at room temperature for 1 h (Uttamsingh et al., 1998). The reaction mixture was acidified to pH 3 with 1M H3PO4 and extracted with ethyl acetate (3 x 10 ml). The crude product from the extracts was purified by column chromatography (2% MeOH-ethyl acetate). The fractions containing the pure product were combined and evaporated to the consistency of thick syrup, 1.7 g (81%). The 1H NMR (nuclear magnetic resonance) spectra in CDCl3 were acquired using a Bruker DPX-300 NMR Spectrometer (Bruker Biospin Corp., Billerica, MA) and agreed with the published spectrum (Uttamsingh et al., 1998); 13C NMR(D2O) 
13.1, 22.5, 22.6, 33.6, 34.5, 52.0, 171.6, and 172.7; GC–MS (AcPrCys as 4-t-butyldimethylsiloxy-3-pentene-2-one (tBDMS) derivative) m/z 319 (10, M-15), 262 (60, M-57), 203 (100, 262—NH2COCH3). The above reaction was repeated on a 2 mmol scale using 1-BP-d7 to obtain d7-AcPrCys; GC-MS m/z 326 (1, M-15), 269 (30, M-57), 210 (100, 269—NH2COCH3).
PrCys and S-(heptadeuteriopropyl)cysteine.
Cysteine (1.75 g, 10 mmol) was dissolved in 2M NaOH (10 ml) and stirred with 1-BP (1 ml, 1 mmol) in ethanol (5 ml) for 3 h. The pH was lowered to 3 with 2M HCl and the solid was filtered, washed with ice-cold water and ethanol, and dried; 1.2 g; mp 207°C–208°C (Nishimura and Mizutani, 1974). The deuterio analog, d7-PrCys (MS, 171 (M + 1)), was similarly prepared using 1-BP-d7.
Diazomethane.
Diazomethane was synthesized according to a previously described method (Hudlicky, 1980).
Animals and Exposures
All experiments were conducted in accordance with Japanese law and the Guide of Animal Experimentation, Nagoya University Graduate School of Medicine. Male Wistar rats (specific pathogen free, 9 weeks old) were purchased from Shizuoka Laboratory Center (Shizuoka, Japan) and acclimated for 1 week in a temperature and humidity controlled room on a diurnal light cycle. Food and water were given ad libitum, except when food was removed during the exposure period to avoid contamination with 1-BP. The inhalation exposure system used in the present study has been described (Ichihara et al., 2000a; Takeuchi et al., 1989). In brief, the regulated volume of 1-BP was measured every 10 s by GC and controlled to within ± 5% of the target concentration by means of a personal computer.
In experiment 1, 32 rats were divided into four groups (n = 8) and were exposed to either 1-BP at 50, 200, and 800 ppm, or fresh filtered air for 8 h/day (10:00–18:00 h) for 2 weeks. On the first exposure day, rats were placed into individual metabolic cages inside the inhalation chambers and urine was collected during the entire 8 h of exposure and for 1-h postexposure. The urine was centrifuged for 10 min at 4000 x g and the supernatant stored at –80°C for AcPrCys determination. At the end of the 14-day exposure, the rats were decapitated and blood was collected by heparinized funnel, and the spinal cords (C2-T12) were removed with cold isotonic phosphate buffered saline immediately postmortem and frozen at –80°C.
In experiment 2, 24 rats were divided into two groups (n = 12) and exposed to either 50 ppm 1-BP or fresh filtered air for 8 h/day (10:00–18:00 h) 5 days/week for 4 weeks. Urine from the 1-BP exposure group was collected overnight for 15 h in metabolic cages before the first and after the fifth exposure day (19:00–10:00 h) of each week. Urine samples were centrifuged and stored at –80°C. Blood (0.5 ml) was collected from the 1-BP exposure group in a heparinized syringe from the external jugular vein under ketamine anesthesia (100 mg/kg ip) after the fifth day of exposure of weeks 1, 2, and 3. At the end of the fourth exposure week, six rats from both the control and exposure group were decapitated and blood was collected by heparinized funnel. In addition, urine samples were collected nightly from the remaining 1-BP exposure group rats for 7 more days; and the remaining six controls and five exposed animals (one rat from the exposure group died unexpectedly during week 4 of the experiment) were decapitated and blood collected 8 days postexposure.
Human Exposures
The study design was approved by the ethical committee of Nagoya University Graduate School of Medicine. Signed informed consent was obtained from each worker for all interviews and samples obtained according to the Declaration of Helsinki (World Medical Association, 2002) and samples analyzed as authorized by the Vanderbilt University Institutional Review Board for human subjects. One blood sample was obtained from each of 26 female workers in a 1-BP production factory located in Yixing, Jiangsu Province, China, who were exposed to 1-BP by inhalation and possible skin contact (Ichihara et al., 2004a). Postshift urine sample procurement and personal exposure monitoring were also performed (n = 48). The workers worked in two shifts from 7:00 to 19:00 and 19:00 to 7:00. Blood samples were handled according to the method described in the next section. Individual exposure levels during work shifts were evaluated with passive samplers (Sibata Scientific Technology Ltd., Tokyo, Japan) using the method described previously by Ichihara et al. (2004a). A passive sampler was attached to each worker during one 12-h shift and was collected immediately after the shift and kept in a separate sealed bag at 4°C until analysis. The adsorbed solvent in the sampler was analyzed 2 weeks after collection. For analysis, activated charcoal particles were extracted with 2 ml of carbon disulfide and the solution was injected into a GC with an electron ionization detector GCD system G1800A (Hewlett Packard, Palo Alto, CA). 1-BP was quantified by the selected ion mode. The time weighted average (TWA) concentration of each solvent was calculated based on the formula: TWA = µg adsorbed solvent/sampling rate (µg/ppm x min) x sampling time (min). The value of 0.134 was used for the sampling rate of 1-BP, which was determined by the diffusion cell method. Individual exposure levels ranged from 0.34 to 49.2 ppm for blood samples and from 0 to 170.54 ppm for urine samples. Blood was also obtained from age-matched control workers (n = 32) from a beer factory in China for globin analysis.
Globin Isolation from Rats and Humans
The blood samples from both the rats and humans were centrifuged to remove plasma, the red cells washed twice with 5mM phosphate buffer, containing 150mM NaCl, pH 7.4, then stored at –80°C. After thawing, 0.4 ml of hypotonic 5mM phosphate buffer, pH 7.4 was added to 0.3 ml of red cells, mixed, and centrifuged at 10,000 x g for 12 min. The resulting hemolysate supernatant was added to 100 µl of 1M ascorbic acid; and the resulting solution was added drop wise to 5 ml of 2.5% oxalic acid in acetone. Globin was allowed to precipitate on ice for 15 min and then centrifuged at 10,000 x g for 10 min at 4°C and washed in 5 ml of ice-cold acetone. The pellet containing crude globin was dried under a stream of N2 and stored at –80°C.
GC/MS/MS Measurement of PrCys on Globin
Rat globin samples (4–5 mg) were hydrolyzed at 109°C for 18 h with 6N HCl using the Waters PicoTag (Milford, MA) to release PrCys. The acid was removed with a Savant SpeedVac and coevaporated with ethanol (3 x 200 µl). The residue was mixed with the internal standard (5 µl (controls and 50 ppm exposures), 10 µl (200 and 800 ppm exposures) of 100µM d7-PrCys), neutralized with freshly distilled triethylamine (10–15 µl to achieve pH 8.0–9.0), and dissolved in 200 µl of derivatizing buffer (50mM triethylammonium bicarbonate in 1:1 water–acetonitrile, pH 10). The derivatizing agent, PyITC (50 µl of 50mM solution in acetonitrile) was added; and the mixture was incubated with slow agitation at 37°C for 1 h and evaporated completely. The residue was dissolved in 100 µl of 10mM HCl and filtered using 0.22 µm Spin-X filters. For LC/MS/MS analysis, the derivatized amino acids were separated on a Waters Xterra MS-C18 column (2.1 x 100 mm, 3.5 µm) at a flow rate of 0.2 ml/m using a Waters Alliance 2690 Separations Module (Milford, MA) connected to a Finnigan TSQ-7000 (San Jose, CA) mass spectrometer; and MAGIC 2.4.13 software (Michrom BioResources, Auburn, CA) was used for data acquisition. The gradient elution was from 100% A (10% methanol in 5mM formic acid) to 40% A and 60% B (100% methanol containing 5mM formic acid) in 20 min and was kept at 60% B for 5 more minutes. Optimization of the molecular ion and the daughter ions was performed by syringe infusion using a standard solution of derivatized PrCys. Positive ion electrospray ionization was used to obtain the parent ions in quadrapole 1, selected parent ions were collided with an inert gas in quadrapole 2, and the resulting daughter ions were detected in quadrapole 3. The molecular ions 300 and 307 were selected, respectively, for PrCys–pyridinylthiocarbonylcysteine (PyTC) and d7-PrCys–PyTC in the first MS; and after cid, the daughter ion at 137 was used for selected reaction monitoring (SRM) (Fig. 1A). A standard curve for calculating the amount of PrCys per milligram of globin was obtained by injecting known mixtures of analyte and internal standard, acquiring peak areas for the daughter ion for both molecular ions and plotting their peak area ratio against the mole ratios. The limit of detection by LC/MS/MS of pure PyTC–PrCys was determined to be 0.2 pmol, which was the lowest injected amount giving a peak area with a signal to noise ratio greater than 5. The limit of detection of PrCys protein adduct was determined by taking protein samples containing differing amounts of synthetic PrCys and subjecting them to hydrolysis, derivatization, and analysis by LC/MS/MS. By this latter method, the lowest amount of PrCys that produced a peak, using the daughter ion at 137, with a signal to noise ratio greater than 5 was 2.5 pmol.
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For the analysis of human globins for PrCys, 4–6 mg of globin was analyzed and several changes were made in the above protocol. After finding that the PrCys analyte was nondetectable when human globin was hydrolyzed for the18 h used for the rat globins, it was determined through numerous trials that 6 h was optimal for hydrolysis and recovery of the analyte. Internal standard (d7-PrCys) was added at a lower amount and concentration (5 µl of 20µM) to provide a suitable mole ratio to the smaller amounts of PrCys analyte found on the human samples. The more sensitive ThermoFinnigan (San Jose, CA) TSQ® Quantum mass spectrometer equipped with the Surveyor® Autosampler and MS Pump (San Jose, CA) was used for analyte detection. Syringe infusion of a standard derivatized solution of PrCys was used to optimize the conditions, with the system automatically performing the optimization. In addition, the daughter ion 137 was found to have background peaks interfering with accurate quantitation, so the daughter ion 164 that produced a lower background in globin protein samples was used for SRM (Fig. 1A).
Neurofilament Isolation and LC/MS/MS Analysis
After thawing the collected rat spinal cords, neurofilaments were isolated as previously described (Liem, 1982). Following removal of the meninges, the spinal cords were homogenized in a solution (2 ml/g spinal cord) containing 10mM phosphate buffer, pH 6.8, 1mM ethylenediaminetetraacetic acid, 0.1mM NaCl, 1mM phenyl methyl sulfonyl fluoride, 1µg/ml leupeptin, 1% Triton-X, and 0.85M sucrose and stirred overnight at 4°C in the homogenizing solution. A pellet containing neurofilament protein was obtained by centrifugation (90,000 x g for 30 min), which was washed with the same solution minus sucrose and Triton-X and stored in this solution at –80°C. Protein determinations were performed on the neurofilament solutions using the Bradford Assay; and aliquots corresponding to 
1 mg of protein were freeze dried. The neurofilament samples were then subjected to the same procedure as described for globin above for PrCys and d7-PrCys separation and quantitation.
LC/MS/MS Measurement of Urinary AcPrCys
A 100 µl aliquot of rat urine (800 and 200 ppm exposed samples were diluted 100 times, 50 ppm samples were diluted 10 times, with distilled H2O) was spiked with internal standard (10 µl of 100µM solution of d7-AcPrCys), acidified with 100 µl of 0.25N HCl, and loaded onto an Oasis-HLB cartridge (30 mg) that had been conditioned with methanol (2 x 0.5 ml) and 10mM HCl (2 x 0.5 ml). The cartridge was washed with 10mM HCl (2 x 0.5 ml) and 10mM HCl in 20% methanol–water (2 x 0.5 ml), and dried. Both the AcPrCys and standard were eluted with methanol (2 x 0.5 ml), dried under a stream of nitrogen, and esterified with excess diazomethane in ether. After the removal of ether, the residue was taken up in 100 µl of Solvent A (10% acetonitrile in 5mM formic acid) and used for chromatographic analysis. The column (Waters Xterra MS-C18 3.5 µm, 2.1 x 100 mm) was eluted with a gradient going from 100% Solvent A to 100% B (100% acetonitrile with 5mM formic acid) in 10 min at a flow rate of 0.2 ml/min. The latter composition was held for 5 min before reverting to A in 1 min. This was accomplished using a Waters Alliance 2690 Separations Module (Milford, MA) connected to a Finnigan TSQ-7000 (San Jose, CA) mass spectrometer and MAGIC 2.4.13 software (Michrom BioResources, Auburn, CA) for data acquisition. Conditions were optimized by syringe injecting a standard solution of the methyl ester of AcPrCys and manually varying the parameters to obtain optimum values. Under these conditions methyl esters of AcPrCys and d7-AcPrCys were eluted at 9.8–9.9 min. Parent ion with m/z of 220 (227 for d7 analog) along with daughter ions at 161 (168) and 118 (125) were used for SRM (see Fig. 1B). The levels of AcPrCys and d7-AcPrCys were quantified with a standard curve that was generated by extracting 100 µl of control urine spiked with 2, 4, 8, 10, 15, and 20 µl of 100µM AcPrCys and 10 µl of 100µM deuterated internal standard. The measured metabolite value was normalized to the level of urine creatinine determined with the Sigma Creatinine Kit 555-A.
GC-MS Measurement of AcPrCys in Rat and Human Urine
Each rat urine (800 ppm exposed urines were diluted 100 times, 200 ppm, 10 times) sample (100 µl) was mixed with 5 µl of 1mM d7-AcPrCys (800 ppm samples were mixed with 10 µl), acidified with two drops of 6M HCl, and extracted with an equal volume of ethyl acetate twice. The extracts were combined and dried (Na2SO4), evaporated with a centrifuging concentrator at room temperature, and derivatized with tBDMS (Ohie et al., 2000) and injected on a GC/MS (Hewlett Packard 5890). The (M-57)+ ions of the tBDMS derivatives of AcPrCys and d7-AcPrCys, 262 and 269 respectively, were followed by single ion monitoring (SIM) (Fig. 1C). The levels of AcPrCys–tBDMS and d7-AcPrCys–tBDMS were quantified using a standard curve that was generated by extracting and derivatizing 100 µl of control urine spiked with AcPrCys at 10, 20, 50, 100, 200, 300, or 500µM and 10 µl of 1mM internal standard and plotting peak area ratios against the mole ratios. The measured metabolite value was normalized to the level of urine creatinine determined with the Sigma Creatinine Kit 555-A.
Human urine samples were processed without dilution and analyzed using the same procedure as the above control rat urine samples.
Statistical Analysis
One-way analysis of variance, Tukey–Kramer's multiple comparisons test, and the unpaired one-tailed t-test were performed using InStat (GraphPad Software, Inc.). Statistical significance was taken to be p < 0.05 unless otherwise noted. Linear fit was performed using KaleidaGraph (version 3.6, Synergy Software).
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RESULTS |
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Rat Studies
LC/MS/MS analysis of globin and neurofilament preparations.
To determine the levels of PrCys adducts produced on hemoglobin and within the central nervous system, globin and neurofilament preparations from animals exposed for 2 weeks at 0, 50, 200, and 800 ppm 1-BP were analyzed by LC/MS/MS. The levels of PrCys adduct on isolated globin showed a linear dose-dependent increase (R2 = 0.997) as shown in Figure 2A. Globin isolated from rats dosed with 1-BP at 50 ppm over 4 weeks (Fig. 2B) showed a linear accumulation (R2 = 0.987) of PrCys adduct of 32.9 pmol/mg globin/week over the course of treatment, dropping by 25.4% (an elimination of 28.1 pmol/mg globin) 1 week after discontinuing treatment.
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25% during 1-week postexposure (n = 12 controls and week 1; n = 11 week 2 and week 3; n = 6 week 4; n = 5 week 5). **p < 0.01 relative to all groups except week 5 group (1-week postexposure), +p < 0.01 relative to all other groups. (C) 1-BP exposure for 2 weeks at 0, 50, 200, and 800 ppm by inhalation produced a linear dose response for PrCys adducts on neurofilament protein (n = 8). *p < 0.01 relative to controls, 50, and 200 ppm groups.LC/MS/MS measurement of PrCys adduct on neurofilaments (Fig. 2C) followed a linear dose–response (R2 = 0.983) similar to globin, although the amounts were only 2–5% of those measured on globin.
Urinary AcPrCys Measurement
As shown in Figure 3A, AcPrCys in urine, obtained during the first day of exposure in the dose–response experiment, determined by LC/MS/MS and GC/MS showed a similar linear dose response (y = 19.263x, R2 = 0.999 for LC/MS/MS and y = 19.245x, R2 = 0.957 for GC/MS) for both methods.
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Measurement of AcPrCys by LC/MS/MS over 4 weeks at the 50 ppm dose showed that the level of urinary AcPrCys increased from undetectable levels prior to starting, to over 30 nmol/mg creatinine at the end of the first week. In subsequent weeks, a residual level of about 4 nmol/mg creatinine was detected on Monday prior to starting exposures, with similar overnight end of week levels of 24–38 nmol/mg creatinine observed. (Fig. 3B). Urinary AcPrCys decreased from 38 nmol/mg creatinine to approximately 4 nmol/mg creatinine during the first 2 days after the last exposure of week 4 (Fig. 3C), and then decreased to 2.5 nmol/mg creatinine during the next 6 days.
Human Studies
LC/MS/MS analysis of globin and GC/MS analysis of urinary AcPrCys.
In globin samples obtained from the 1-BP exposed workers, a peak could be identified between 15.5 and 16 min corresponding to the 164 m/z daughter of PrCys (Fig. 4). As seen in Figure 5A, the level of PrCys adduct as determined by LC/MS/MS on human globin samples was much less than levels detected in the rat studies. At the exposure levels seen in these workers (0.32–49.2 ppm) there was a significant increase in PrCys adducts present in the globin of 1-BP factory workers over the control workers. Urinary AcPrCys, as determined by GC/MS, increased for the most part with increasing exposure levels measured by passive sampling (Fig. 5B).
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DISCUSSION |
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The neurotoxic effects of 1-BP may, at least in part, be mediated through its ability to covalently bind to the sulfhydryl groups in the nervous system. This can result either from the direct addition of 1-BP to sulfhydryl functions to generate S-propyl cysteine adducts as observed here (Fig. 6) for proteins and previously reported for glutathione (Jones and Walsh, 1979
) or from reactive metabolites such as 1-bromo-2-propanol metabolites that can add to sulfhydryl functions (Barnsley et al., 1966). Accordingly, previous publications have shown that inhalation exposure to 1-BP decreased cytosolic nonprotein sulfhydryl groups and glutathione concentrations of rat cerebrum and cerebellum after 7 days of exposure (Wang et al., 2002) and decreased both protein-bound and nonprotein sulfhydryl groups and total glutathione in cerebrum, cerebellum and brainstem in rats after 12 weeks of exposure (Wang et al., 2003). The formation of PrCys adduct on rat spinal cord protein in the present study presents a potential mechanism to explain the observed decrease in sulfhydryl groups in those previous studies.
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The PrCys adducts on neurofilament proteins and globin were identified by SRM LC/MS/MS after derivatizing with PyITC. PyITC was used instead of the more commonly used phenylisothiocyanate due to the ability of the pyridinyl ring to form a positive ion that is more easily detectable by LC/MS/MS, thus increasing sensitivity. The studies presented in this paper demonstrated the utility of this LC/MS/MS method and exhibited linearity over the measured range of 0 to approximately 2000 pmol PrCys/mg globin, and the limit of detection was adequate to detect the human exposures in this factory when approximately 5 mg of globin was used for analysis. To enable analysis of lower exposures, more globin could be analyzed with this method. One important finding from the LC/MS/MS analyses was that due to the much smaller amounts of adduct present on human globin as compared to the rats in these experiments, the presence of interfering peaks using daughter ion at m/z 137 made measurement unreliable. Even though the daughter ion at m/z 164 was less abundant, it was chosen for LC/MS/MS analysis in humans due to its greater signal to noise in biological samples at low adduct levels.
The first rat study presented here demonstrated a linear dose-dependent rise of PrCys adduct on globin over the exposure range of 0–800 ppm over a 2-week time period; and the second rat study illustrated the linear cumulative response of globin adduct over a 4-week period at 50 ppm, demonstrating its ability to integrate internal exposures to 1-BP over this exposure period. In addition, 75% of the adducts were still detected 1 week after the end of the 4-week exposure, consistent with its chemical stability. The exposures measured from the 1-BP factory workers in this study from whom globin was obtained ranged from 0.34 to 49.2 ppm. Positive findings in this exposure range support the utility of this assay and suggest that this method is suitable for monitoring exposures at nontoxic levels of exposure. Previous investigations of neurologic disease in workers exposed to 1-BP have reported exposure levels of 60–261 ppm in a cushion company (Ichihara et al., 2002), 91–176 ppm in another cushion company (Majersik et al., 2007), and a geometric mean of 1.42 ppm with a maximum of 27.8 ppm in workers engaged in cleaning and painting metal surfaces with preparations containing 1-BP (Kawai et al., 2001). In another study investigating DNA damage in leukocytes of workers from two other cushion factories, TWA concentrations of 1-BP ranged from 0.2 to 271 ppm at one facility and from 4 to 27 ppm at another facility (Toraason et al., 2006). Individual 1-BP worker globin adduct levels were measured here but not compared to 1-BP passive sampler levels in this study, as information about previous exposure conditions for each worker was not known. Further studies are needed to assess the relationship of globin adduct levels to neurologic abnormalities in humans in the event that these may be predicted in a dose-dependent manner. In addition, further studies to measure adduct elimination in human exposed populations could be designed in an attempt to estimate exposure levels in those individuals seeking medical treatment for neuropathies who are not currently at the work site.
Past studies (Garner, 2006; Ichihara, 2005; Jones and Walsh, 1979) have helped to elucidate the metabolism of 1-BP in mice and rats. Previous studies in rats and humans (B'Hymer and Cheever, 2004; Garner et al., 2006; Ishidao et al., 2002; Jones and Walsh, 1979; Kawai et al., 2001) have identified urinary metabolites including bromide ion, N-acetyl-S-propylcysteine-S-oxide, N-acetyl-S(2-hydroxypropyl)cysteine, N-acetyl-S(3-hydroxypropyl)cysteine N-acetyl-3-(propylsulfinyl)alanine, 1-bromo-2-hydroxypropane-o-glucuronide, N-acetyl-S(2-carboxyethyl)cysteine N-acetyl-S(2-oxopropyl)cysteine, N-acetyl-3-((2-oxopropyl)sulfinyl), 3-bromopropionic acid, and 1-BP with potential for use as biomarkers of 1-BP exposure. However, few studies have examined dose response, accumulation, or elimination data of these biomarkers in rats and at occupationally significant exposure levels. AcPrCys detected in the urine of rats and humans is proposed to form following conjugation of glutathione with 1-BP with further metabolism to the mercapturate, AcPrCys. In the rat studies presented here, AcPrCys determined by GC/MS correlated very well with determinations made by LC/MS/MS and provides for another available method for urine AcPrCys analysis. Linearity of both methods was demonstrated in rats over a range of 0–16,000 nmol/mg creatinine.
Urinary levels of AcPrCys followed a linear dose response over the 0- to 800-ppm exposures examined in rats. The generation of AcPrCys can occur from the direct reaction of 1-BP with glutathione; and this dose–response relationship may not be influenced by factors influencing metabolism of 1-BP, e.g., gender, species. It is possible that glutathione-S-transferase may catalyze the conjugation of 1-BP to glutathione; and if this is true, individuals may have isoforms that influence the rate of elimination. The rapid elimination of AcPrCys immediately following cessation of exposure may complicate the use of this marker for quantifying exposures and probably accounts for the difference in urinary AcPrCys observed for the two different sampling methods used for the acute and subacute 50 ppm exposures in rats. This rapid elimination may also explain the lower correlation of AcPrCys to exposure level observed in the human samples; however, the exposure levels of those individuals in days prior to urine collection was also not determined and may have affected the results. Nevertheless, the levels of AcPrCys detected and the persistence of this metabolite in urine support its utility for detecting 1-BP exposure at relevant levels and signaling excessive exposure levels.
Biomarkers that are produced in a dose-dependent manner and can reflect acute, subacute and chronic exposures that can be reliably assayed are needed to protect the vulnerable human population exposed to 1-BP in the workplace. The present study demonstrates the ability of 1-BP to covalently modify proteins within the nervous system as a potential contributing mechanism for 1-BP observed neurological effects, and supports the potential of 1-BP-mediated S-propyl modifications of sulfhydryl functions and urinary AcPrCys to serve as biomarkers of exposure. In this case of protein modification, globin PrCys adducts showed promise as markers with the ability to integrate exposures over the period of a month with adequate sensitivity for analysis of occupational exposures. The urinary metabolite AcPrCys provides for greater ease of sample procurement with suitable sensitivity for assessing workplace exposures and an extended elimination phase. To utilize these markers, further studies are required to delineate dose–response relationships in humans and guidelines for exposure levels in humans established. A better understanding of the underlying mechanism of 1-BP's adverse biological effects will facilitate our understanding of 1-BP toxicity, establishment of informed exposure level guidelines, and development of appropriate biomarkers.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONFUNDINGREFERENCES |
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National Institute of Environmental Health Sciences grant (ES06387-8); Center of Molecular Toxicology Grant (P30 ES00267); and Japan Society for the Promotion of Science Grant (16390169).
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NOTES |
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This paper was presented in part at the 46th Annual Society of Toxicology Meeting in Charlotte, NC, in March 2007.
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ACKNOWLEDGMENTS |
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LC/MS/MS analysis was performed with the assistance of Lisa Manier of the Mass Spectrometry Research Center at Vanderbilt University.
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