Acrylamide: A Comparison of Metabolism and Hemoglobin Adducts in Rodents following Dermal, Intraperitoneal, Oral, or Inhalation Exposure

Susan C. J. Sumner1, Carla C. Williams, Rodney W. Snyder2, Wojciech L. Krol3, Bahman Asgharian and Timothy R. Fennell4

CIIT Centers for Health Research, 6 Davis Drive, Research Triangle Park, North Carolina 27709-2137

Received May 9, 2003; accepted June 30, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acrylamide (AM), which is used to manufacture polymers, is carcinogenic and a reproductive and neurological toxicant. The objective of this study was to compare the metabolism of AM administered orally (po), dermally, intraperitoneally (ip), or by inhalation, and to measure the hemoglobin adducts produced. Rats and mice were exposed to 2.9 ppm [1,2,3-13C] and [2,3-14C]AM for 6 h. [2,3-14C]AM (162 mg/kg) or [1,2,3-13C]AM (13 8 mg/kg) in water was administered dermally to rats for 24 h, and [1,2,3-13C]AM was administered ip (47 mg/kg). Urine and feces were collected for 24 h. Urine was the major elimination route in rats (ip, 62% and po, 53% of the dose; dermal, 44% of the absorbed dose; inhalation, 31% of the recovered radioactivity) and mice (inhalation, 27% of the recovered radioactivity). Signals in the 13C-NMR spectra of urine were assigned to previously identified metabolites derived from AM glutathione conjugation (AM-GSH) and conversion to glycidamide (GA). AM-GSH was a major metabolic route in rats accounting for 69% (ip), 71% (po), 52% (dermal), and 64% (inhalation). In mice, AM-GSH accounted for only 27% (inhalation) of the total urinary metabolites. The remaining urinary metabolites were derived from GA. Valine hemoglobin adducts of AM and GA were characterized using liquid chromatography-mass spectrometry. The ratio of AM to GA adducts paralleled the flux through pathways based on urinary metabolites. This study demonstrates marked species differences in the metabolism and internal dose (Hb-adducts) of AM following inhalation exposure and marked differences in uptake comparing dermal with po and ip administration.

Key Words: acrylamide; glycidamide; metabolism; dermal; intraperitoneal; inhalation; hemoglobin adducts.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acrylamide (AM) is used in the chemical industry to manufacture polymers, as a grouting agent, and in the production of specialty monomers (IARC, 1994Go). Humans can be exposed to AM by ingestion, inhalation, and skin contact (Dearfield et al., 1995Go; IARC, 1994Go). Recent investigations have indicated that AM is formed in fried foods (Tareke et al., 2002Go). Humans exposed to AM over prolonged periods of time can experience peripheral neuropathy, characterized by a dying back of elongate nerve cell axons (Miller and Spencer, 1985Go; Spencer and Schaumberg, 1974Go). AM is neurotoxic, genotoxic, and carcinogenic in rodents (Bull et al., 1984aGo,bGo; Damjanov and Friedman, 1998Go; Dearfield et al., 1995Go; IARC, 1994Go; Tyl et al., 2000Go). The majority of short-term studies of AM effects have been conducted with gavage or ip administration. Studies of subchronic and chronic AM toxicity in rodents have been conducted primarily after po administration either in the drinking water or diet. There is little toxicology information following dermal or inhalation exposure to AM. Chromosomal damage in germ cells of male mice following dermal administration of AM has been reported (Gutierrez-Espeleta et al., 1992Go). AM administered dermally had a lower activity for skin tumor initiation than AM administered po (Bull et al., 1984aGo). This current research addresses differences in metabolism and internal dose of AM associated with route of administration.

AM is rapidly removed from the blood stream following po and iv administration to rodents (Edwards, 1975Go; Miller et al., 1982Go). Following administration of 14C AM (Hashimoto and Aldridge, 1970Go; Miller et al., 1982Go) or 13C AM (Sumner et al., 1992Go) to rats and mice, 40 to 70% of the dose was excreted in urine by 24 h. Direct glutathione (GSH) conjugation with AM results in the urinary excretion of N-acetyl-S-(3-amino-3-oxopropyl)cysteine and S-(3-amino-3-oxopropyl)cysteine (Miller et al., 1982Go; Sumner et al., 1992Go, 1997Go). The epoxide glycidamide (GA) has been detected in the urine of rodents exposed to AM (Sumner et al., 1992Go). GA reacts with DNA (Segerback et al., 1995Go) and is mutagenic (Barfknecht et al., 1988Go; Hashimoto and Tanii, 1985Go).

The uptake, distribution, and metabolism of AM following dermal or inhalation exposure has received little attention. Radioactivity derived from 14C-labeled AM administered to Sencar and BALB/c mice by po or dermal routes was compared in lung, liver, stomach, and skin. Little difference was found between tissues, with the exception of skin, which contained much higher levels of radioacitivity with dermal exposure (Carlson and Weaver, 1985Go).

GA, its hydrolysis product (2,3-dihydroxypropionamide), and products derived from GSH conjugation with GA, including N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine and N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine, are excreted in urine of AM-exposed rats and mice (Sumner et al., 1992Go, 1997Go). Mice devoid of cytochrome P4502E1 do not excrete GA or GA-derived metabolites following a 50 mg/kg po AM dose (Sumner et al., 1999Go). These findings indicate that P4502E1 is the major isozyme involved in the in vivo metabolism of AM and that other cytochromes P450 do not metabolize AM in the absence of P4502E1.

Hemoglobin adducts from both AM and GA have been measured in rats administered AM (Bergmark et al., 1991Go), in people occupationally exposed to AM, and in smokers (Bergmark, 1997Go; Bergmark et al., 1993Go; Calleman et al., 1994Go). Adducts formed by reaction of chemicals and their metabolites in hemoglobin provide a means of assessing exposure and of measuring internal dose. Both AM and GA react with the N-terminal valine residue of globin. AM forms N-(2-carbamoylethyl)valine (AAVal), and GA forms N-(2-carbamoyl-2-hydroxyethyl)valine and N-(1-carbamoyl-2-hydroxyethyl)valine (Bergmark, 1997Go; Bergmark et al., 1993Go; Calleman et al., 1994Go). These adducts can be analyzed by the modified Edman reaction, in which adducted valine residues are selectively cleaved on reaction with pentafluorophenylisothiocyanate. The resulting pentafluorophenylthiohydantoin products are extracted and analyzed by gas chromatography/mass spectrometry. A new sensitive and more rapid method for analysis of AM and GA hemoglobin adducts has been developed using liquid chromatography with tandem mass spectrometry (Fennell et al., 2003Go).

The objective of this study was to compare the extent of metabolism of AM administered dermally, ip, or by inhalation and to measure the hemoglobin adducts produced. 13C-NMR was used to detect and quantitate metabolites in urine of rats following inhalation exposure, dermal application, or ip injection of [1,2,3–13C]AM. The percentage of metabolism that occurred via P450 oxidation of AM to GA was compared for each exposure route. The distribution of [14C]AM was established following dermal application and inhalation exposure. AAVal and GAVal formed in hemoglobin were measured to evaluate the effect of exposure route on uptake and metabolism of AM to GA. AAVal and GAVal provide a measure of the area under the curve of AM and GA, respectively, in blood. The ratio of AM to GA provides a measure of the relative internal dose of AM and GA.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
[2,3–14C]AM (99% chemical purity and a specific activity of 5 mCi/mmol) was obtained from American Radiolabeled Chemical, Inc. (St. Louis, MO). The unlabeled AM (99.9% chemical purity) was obtained from Bio-Rad Laboratories (Hercules, CA). The 13C-NMR spectrum for unlabeled AM had singlets at 129 ppm (H2C), 130 ppm (CH), and 171 ppm (CO). The 1H-NMR spectrum had multiplets at 5.83 ppm (H2C) and 6.35 ppm (CH) respectively. The [1,2,3–13C]AM used in the inhalation study was obtained from Cambridge Isotope Labs (Andover, MA). The [1,2,3–13C]AM used in the dermal study was purchased from ISOTEC Inc. (Miamisburg, OH). Both contained a 99% isotopic enrichment and >98% chemical purity. The 13C -NMR spectrum of the [1,2,3–13C]AM had multiplets at 126–132 ppm and a doublet (Jcc = 51 Hz) at 171 ppm, consistent with the 13C -labeled carbons for the vinyl group (C = C) and carbonyl group (C = O), respectively. 1,4-Dioxane (>99%) was purchased from Aldrich Chemical Company (St. Louis, MO). N-(2-Carbamoylethyl)valine-leu-anilide was obtained from Bachem (King of Prussia, PA). Phenylisothiocyanate was obtained from Aldrich (Milwaukee, WI). Valine and valine-13C5 were obtained from Sigma (St. Louis, MO) and Isotec Inc. (Miamisburg, OH), respectively.

Glycidamide was synthesized as previously reported (Fennell et al., 2003Go). The syntheses of the adduct standards N-(2-carbamoylethyl)valine (AAVal), N-(2-carbamoylethyl)valine -13C5 (AAVal-13C5), N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal), and N-(2-carbamoyl-2-hydroxyethyl)valine -13C5 (GAVal-13C5), and their respective phenylthiohydantoin derivatives were reported in Fennell et al.(2003)Go.

Animals.
Male F344 rats and male B6C3F1 mice were purchased from Charles River Laboratories (Raleigh, NC) and acclimated for at least 13 days. Rats and mice were housed in microisolator cages containing Alpha Dri direct contact bedding. At the time of dosing, rats and mice were 9–10 weeks old. They were supplied food (NIH-07 diet, Ziegler Brothers) and reverse-osmosis water ad libitum (except during inhalation exposure) and maintained on a 12-h light-dark cycle (0700–1900 h for light phase) at a temperature of 64–79°F and relative humidity of 30–70%. All animal care and procedures were conducted according to the Guide for the Care and Use of Laboratory Animals, National Institutes of Health (1985)Go.

Animal exposures.
Three separate groups of exposures were conducted: (1) dermal, po, and ip administration of [1,2,3-13C]AM to rats, (2) dermal administration of [2,3-14C] AM to rats, and (3) inhalation exposure of rats and mice to a mixture of [1,2,3-13C]AM and [2,3-14C] AM.

Selection of dose and sample collection period.
An ip dose of 50 mg/kg was selected to facilitate direct comparison with results obtained following a 50 mg/kg gavage dose of [1,2,3-13C]AM to male F344 rats (Sumner et al., 1992Go). A 150 mg/kg dermal application was selected based on in vitro studies indicating that up to 30% of applied AM is absorbed (Marty, 1998Go, cited with permission from TEGEWA). The maximum achievable nose-only inhalation exposure concentration was 5.6 ppm for unlabeled AM and 2.9 ppm for the mixture of 13C- and 14C-labeled AM. Previous studies with rats or mice administered [14C]AM (Hashimoto and Aldridge, 1970Go; Miller et al., 1982Go) or [13C]AM (Sumner et al., 1992Go, 1997Go, 1999Go) indicated that ~40 to 70% of the dose was excreted in urine within 24 h following exposure.

Dosing
Dermal exposures, po and ip administrations.
A 53 mg/g dosing solution of [14C]AM/AM and a 48 mg/g dosing solution of [1,2,3-13C]AM (both in distilled water) were administered within 1h of preparation. The specific activity of the [14C]AM/AM dosing solution was 273.16 µCi/mmol. Approximately 24 h prior to dermal exposure, fur was clipped from the dorsal area of each rat (~5 x 10 cm), and the area was washed (10% aqueous solution of Ivory detergent followed by distilled water). A Hilltop ChamberTM (2.5 cm2) was applied to the shaved area using SuperglueTM. The [1,2,3-13C]AM dose (138 ± 1.49 mg/kg; 378 ± 14.0 µmol) was delivered under the patch of four rats via a syringe (~3 ml/kg body weight of the 48 mg/g [1,2,3-13C]AM solution). The [14C]AM/AM dose (162 ± 2.70 mg/kg; 507 ± 24 µmol) was delivered to four additional rats. Draize Scale observations were negative (score = 0) after shaving, prior to dermal application, and 15 min (viewing edge of patch) and 24 h after dermal application. Patches were checked for leaks just after dermal application and during the sample collection phase of the study. Four additional rats were administered (ip) the 48 mg/g dosing solution of [1,2,3-13C]AM at ~ 1 ml/kg body weight (actual AM dose was 46.5 ± 3.30 mg/kg; 126 ± 11.6 µmol). At the time of dosing, rats weighed 212–237 g for the [14C]AM dermal study, 195–209 g for the 13C dermal study, and 197–206 g for the ip study. The rats were transferred to glass metabolism cages immediately after dosing.

Oral administration of [1,2,3-13C] acrylamide to rats at a nominal dose of 50 mg/kg was described previously (Fennell et al., 2003Go). The actual dose administered was 59.5 ± 8.0 mg/kg.

Inhalation exposures.
Acrylamide vapor was generated from solid acrylamide in a glass J-tube heated to 75°C in a temperature controlled water bath. Heated nitrogen was passed throught the J-tube, and the resulting acrylamide vapor passed through a column of glass beads. Heated oxygen was passed through an impinger containing water, and was mixed with the acrylamide vapor in nitrogen in a mixing tee. The temperature of the delivery line was monitored using a temperature probe. The exposures were conducted using a modified Cannon nose-only tower (Cannon et al., 1983Go). Heating tape was wrapped around the delivery line, and the top of the exposure tower. The temperature at the exposure port was monitored and controlled at <79°F.

The system for generation of the acrylamide atmosphere was connected to the exposure system with a 3/8'' Teflon tee fitted with a needle sampling port and a Teflon-faced septum, so that samples of the acrylamide atmosphere could be withdrawn for analysis. Exposure concentrations were monitored by two methods, one involving GC with FID detection, and the other by HPLC. The GC method was not calibrated, because no reliable method could be found to generate acrylamide vapor standards, but the method provided a quick useful means to monitor acrylamide concentration stability. Air samples (0.5 ml) were withdrawn at approximately 15-min intervals for analysis by GC using an HP 5890 (Hewlett Packard, Palo Alto, CA) equipped with a splittless injection port and two HP-1 columns in series (5 m x 0.53 mm, 2.65 µm film thickness). HPLC analysis was conducted to determine the vapor concentration of acrylamide (HP1100 Chemstation, Hewlett Packard, Palo Alto, CA). Vapor samples of 25 ml were taken at approximately 30-min intervals. The samples were passed through water (150 µl) at a rate of 5 ml/min, and then analyzed by HPLC using a Beckman Ultrasphere ODS column, (C18, 0.46 x 25 cm, 5 µm) with an Ultrasphere All-Guard cartridge (C18, 7.5 mm x 4.6 mm, 5 µm). Elution was conducted with 100% water at a flow rate of 1.0 ml/min. A calibration curve was constructed using acrylamide standards prepared in water. Detection of acrylamide was conducted by measuring absorbance at 195 nm. The concentration of acrylamide in the air sample was calculated from the concentration of acrylamide in the aqueous solution.

A mixture of [1,2,3-13C]AM (90%) and [2,3-14C]AM (10%) was prepared with a specific activity of 568.65 µCi/mmol. Rats and mice were loaded into nose-only tubes and exposed to the AM vapor on a modified Cannon nose-only tower. For the pilot study, three male rats and two male mice were exposed to ~ 5.6 ppm unlabeled AM for 6 h and transferred to shoebox cages (1/cage) for 24 h. For the definitive inhalation study, eight male rats and eight male mice were exposed to a mixture of 13C and 14C AM. The average 6-h exposure concentration of AM-vapor was 2.90 ± 1.17 ppm. The body weights ranged between 205 and 217 g for rats and between 26 and 34 g for mice. At the end of the 6-h exposure, four male rats and four male mice were immediately sacrificed for the collection of tissues. The additional four male rats and four male mice were transferred to glass metabolism cages.

Sample collection.
Following inhalation exposure, dermal application, or ip administration, four male rats and four male mice (inhalation only) were placed in all-glass metabolism cages (1/cage). Air was drawn through the metabolism cage under negative pressure and passed through charcoal filters on exiting the cage. Urine (over dry ice) and feces were collected for 0–24 h. Exhaled volatiles and 14CO2 (1.0 M potassium hydroxide traps) were collected 0–2, 2–6, and 6–24 h following administration of [14C]AM. At 24 h after [14C]AM dermal application or inhalation exposure, rodents were sacrificed for the collection of blood (CO2, cardiac puncture) and lungs, abdominal fat, subcutaneous fat, thymus (dermal study only), spleen, liver, testes, epididymis, kidneys, brain, stomach, intestines, skin (site of application, dermal study only), skin (nondose site, dermal study only), skin (inhalation study), and carcass. Blood was also collected from rats exposed to [13C]AM. The blood was centrifuged to prepare washed red blood cells for hemoglobin adduct analysis. All samples were stored at ~ -20°C with the exception of urine which was stored at ~ -80°C. Urine volumes (0–24 h) were 6 to 7 ml (rats, [14C]AM, dermal), 6–9 ml (rats, inhalation, [13C 14C]AM), 1 to 2 ml (mice, inhalation, [13C 14C]AM), 8–11 ml (rats, ip, [13C]AM), and 11–14 ml (rats, dermal, [13C]AM).

Sample Evaluation
Distribution.
Red blood cells were separated from plasma by centrifugation at 2000 x g for 20 min. Tissues, blood, plasma, red blood cells, and feces (softened with 1% Triton X-100) were digested in tetraethyl ammonium hydroxide (TEAH), and aliquots were neutralized with concentrated HCl and decolorized with hydrogen peroxide (30% H2O2). Total radioactivity was determined using a Packard 1900 CA Tricarb LA Analyzer after addition of liquid scintillation fluid (EcoLumeTM, ICN, CA). Aliquots of the urine, KOH traps, and cage and nose-only tube washes were analyzed directly by scintillation counting after addition of scintillation fluid. Exhaled volatile [14C]AM equivalents were extracted from charcoal traps using N,N-dimethylformamide (DMF), and aliquots of the extracts were analyzed by scintillation counting.

NMR analysis.
Dioxane (1%) and D2O (99%) were added to urine aliquots. NMR spectra were acquired with a 5-mm dual proton-multinuclear probe on a Varian VXR-300 spectrometer (Palo Alto, CA). Proton decoupled carbon-13 NMR spectra were acquired in the double precision mode with a relaxation delay of 10 s and a 60° pulse width. All 13C -NMR spectra were acquired with >5000 transients. Dioxane was added as an internal reference (66 ppm) and for quantitation. Urea was present at 162.5 ppm.

Metabolites were quantitated by integration against the dioxane internal standard using the following relationship:


The dioxane integral was divided by 4, since it arises from four equivalent carbons, and the ratio of 1.1/99 accounts for the 99% 13C -label on AM relative to the natural abundant (1.1%) 13C-carbons in dioxane. Signals were integrated for at least one carbon in each metabolite, where the multiplet pattern was clearly distinguishable and the integral could be flattened prior to the rise and after the fall of the peak. Metabolites were quantitated to values as low as 0.064 mM (or ~ 6.4 mM 13C-equivalent). AM was observed in urine from rats administered AM by ip injection but was not detected in urine from rats following dermal application or inhalation exposure. AM was not quantitated due to the long relaxation time for the vinyl and carbonyl carbons. NMR has previously been used for quantitation of AM-derived and acrylonitrile-derived urinary metabolites, providing results consistent with other literature using radiolabeled material (Fennell et al., 1991Go; Sumner et al., 1992Go).

Analysis of AAVal and GAVal in hemoglobin.
N-(2-carbamoylethyl)valine (AAVal) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal), formed by reaction of AM and GA respectively with the N-terminal valine residue in hemoglobin, were measured by an LC-MS/MS method (Fennell et al., 2003Go). Globin was isolated from washed red cells (Mowrer et al., 1986Go). Samples were derivatized with phenylisothiocyanate in formamide to form adduct phenylthiohydantoin derivatives in a manner analogous to the modified Edman degradation (Bergmark, 1997Go; Perez et al., 1999Go; Törnqvist et al., 1986Go). Internal standards, AAValPTH-13C5 and GAVal PTH-13C5 were added, and the samples were extracted using a Waters Oasis HLB 3 cc (60 mg) extraction cartridge (Milford, MA). The samples were eluted with methanol, dried, and reconstituted in 100 µl of 50:50 MeOH:H2O (containing 0.1% formic acid). Analysis was conducted using a PE Series 200 HPLC system interfaced to a PE Sciex API 3000 LC-MS with a Turboionspray interface. Chromatography was conducted on a Phenomenex Luna Phenyl-Hexyl Column (50 mm x 2 mm, 3 µm) eluted with 0.1% acetic acid in water and methanol at a flow rate of 350 µl/min, with a gradient of 45–55% methanol in 2.1 min. The elution of adducts was monitored by Multiple Reaction Monitoring (MRM) in the negative ion mode for the following ions:

Quantitation of AAVal was conducted using the ratio of analyte to internal standard, with a calibration curve generated using AAVal-leu-anilide. Quantitation of GAVal was conducted using the ratio of analyte to internal standard.

For samples in which rodents are administered a single dose of [1,2,3–13C]AM to track its metabolism using 13C NMR spectroscopy, the 13C AAVal and 13C GAVal can be distinguished in the negative ion mode from the natural abundance analyte and labeled internal standard since the 13C-containing adduct side chain is lost from the adduct PTH in the collision cell. An additional set of ions is monitored to quantitate the adducts formed:

Statistical analysis.
Statistical analysis was conducted using Instat 2.01 (Graphpad Software, San Diego, CA). Evaluation of species differences in the extent of GA derived metabolites, AAVal, and GAVal, was conducted with Student’s t-test. For evaluation of differences resulting from route of exposure, comparisons of AAVal and GAVal were only made where the dose administered was the same, i.e., with ip and gavage administration of 50 mg acrylamide/kg, with inhalation exposure at 0 and 24 h after exposure, and with two dermal administration studies. Differences in GAVal:AAVal ratio were compared across all treatments with ANOVA using a Tukey Kramer multiple comparisons test for all pairwise comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evaluation of the amount of AM taken up and its disposition following dermal and inhalation exposure was accomplished using 14C labeled AM.

Dermal Application
Following dermal application of 162 mg/kg 14C[AM] to four male F344 rats, radioactivity was recovered from urine, feces, expired air, cage wash, tissues, dermal patch, and wash of the application site. The total recovered radioactivity (including material and wash from the dermal patch dosing site) was within 90% of the applied radioactivity. The absorbed dose was calculated as radioactivity recovered in urine, feces, expired air, cage wash, and tissues (i.e., excludes material from dermal patch and wash of site of application). Following dermal application of 162 mg/kg 14C[AM], the amount of the applied dose that was absorbed was 14, 15, 27, and 30% for the four rats.

The major portion of the dose was excreted in 0–24 h urine (8% of the applied dose or 36% of total absorbed dose) or remained in the body (53% of the absorbed dose) following the 24-h AM-dermal application (Table 1Go). A minor portion of the absorbed dose was recovered in feces (< 1%) or eliminated as organic volatiles (1%) or 14CO2 (2%). Following the 24-h dermal application of [14C]AM, blood cells had the highest relative level (~1 µmol/g tissue) of radioactivity (excluding skin at the dose site) compared with all other tissues. The skin (nondose site, 0.4 µmol/g tissue) and liver, spleen, testes, and kidney (~0.3 µmol/g tissue) had nearly the same levels. Radioactivity (~0.2 µmol/g tissue) was also recovered in the lungs, thymus, brain, and epididymis. Low levels (>0.05 µmol/g tissue) of radioactivity were recovered in fat.


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TABLE 1 The Distribution of [14C]AM Equivalents in Rats during a 162 mg/kg 24-h Dermal Application or in Rats and Mice following a Single 6-h 2.9 ppm Inhalation Exposure
 
Inhalation Exposure
No mortality occurred for male rats or mice exposed to 5.6 ppm AM for 6 h, indicating that the 6-h LC50 for exposure to AM vapor exceeds 5.6 ppm (16.2 mg/m3 or 0.23 µmol/l). The total [14C]AM equivalents recovered from male rats (89 ± 8.9 µmol/kg body weight) were at least 2.8 times lower than [14C]AM equivalents recovered from male mice (245 ± 45 and 401 ± 102 µmol/kg body weight at 0 and 24 h following exposure, respectively). For rats, the radioactivity (µmol) recovered immediately following exposure was not markedly different from that recovered 24 h following exposure (not shown). For mice, the average µmol recovered immediately following exposure (8 ± 1.1 µmol) was not significantly different from the average µmol recovered 24 h following exposure (11 ± 2.7 µmol). The total recovered AM-equivalents 24 h following a 2.9 ppm inhalation exposure to AM (19 µmol) was ~6 times lower that the radioactivity recovered following a 162 mg/kg (112 µmol) dermal application of AM (Table 1Go).

For rats, the major portion of the inhaled dose was excreted in urine (31% of total absorbed dose) or remained in the body (56%) by 24 h following exposure termination (Table 1Go). A minor portion of the absorbed dose was recovered in feces (~3%) or eliminated as organic volatiles and 14CO2 (~2%). A similar distribution of the inhaled dose was determined for mice with 27% in urine, 46% in tissues, 5% in feces, 2% as organic volatiles, and 1% as 14CO2.

Immediately or 24 h following the 6-h inhalation exposure to 2.9 ppm AM, blood cells of rats had the highest relative level (~0.1 µmol/g tissue) of radioactivity compared with all other tissues. Plasma levels were higher immediately following exposure termination (0.03 µmol/g tissue) and reduced 24 h later (0.004 µmol/g tissue). The rank order of relative (µg/g tissue) radioactivity immediately following exposure was blood > testes ~ skin ~ liver ~ kidneys > brain ~ spleen ~ lung ~ epididymis. After 24 h, the rank order of radioactivity was blood > skin > spleen ~ lung ~ liver ~ kidney > brain ~ testes ~ epididymis > fat. Lowest radioactivity levels were observed for fat at either time point. For mice, the rank order of relative radioactivity immediately after exposure was testes, skin, liver, kidney, epididymis, brain, lung, blood, and fat. After 24 h, the rank order was skin, subcutaneous fat, testes, blood, epididymis, liver, lung, spleen, brain, abdominal fat, and kidney.

Metabolites of [1,2,3-13C]AM
For characterization of the involvement of the two main pathways of metabolism of AM, [1,2,3-13C]AM was administered in these studies, either alone or in combination with [2,3-14C]AM. Signals detected in the 13C -NMR spectra of urine from [1,2,3-13C]AM exposed rodents were assigned to the 13C-labeled carbons of AM-derived metabolites based on expected chemical shifts for previously identified metabolites (Fig. 1Go, Sumner et al., 1992Go). The 13C -NMR spectra of urine collected from rodents exposed to [13C]AM contained signals (Fig. 2Go) that possess coupling patterns consistent with the previously assigned metabolites of [13C]AM. These signals are recognized as metabolites of [13C]AM due to multiplet patterns that arise from carbon-carbon coupling between enriched 13C-nuclei. The chemical shifts (center of multiplet pattern) and coupling constants (Jcc) for AM-derived metabolites in exposed rodents are summarized in Table 2Go. Metabolites derived from direct conjugation of AM with glutathione (AM-GSH) include N-acetyl-S-(3-amino-3-oxopropyl)cysteine (metabolite 1) and S-(3-amino-3 oxopropyl)cysteine (metabolite 1'). Diastereomers of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine (metabolite 2,2') and N-acetyl-S-(1-carbamoyl-2- hydroxyethyl)cysteine (metabolite 3, 3') were detected (GSH-GA) and are derived from GSH conjugation with GA (GA, metabolite 4). 2,3-Dihydroxypropionamide and its acid (metabolite 5,5') were definitively assigned only in samples from rats administered [1,2,3-13C]acrylamide by gavage.



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FIG. 1. Proposed metabolism of AM (aCH2 = bCH-cCONH2). The scheme is proposed from metabolites detected in urine of AM-exposed rats and mice. (Adapted from Sumner et al., 1992Go.) Cys = cysteine; N-AcCys = N-acetyl cysteine; GS = glutathione.

 


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FIG. 2. The 13C -NMR spectra of urine obtained from (a) a rat following a 47 mg/kg ip administration of [1,2,3–13C]AM and (b) the expanded region detailing signals for metabolites 1–4.

 

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TABLE 2 Chemical Shifts and Coupling Constants for Metabolites in Urine from Rats and Mice Exposed to [1,2,3-13C]AM
 
Metabolites 1–4 (Fig. 1Go) were detected and quantitated for all rats administered [1,2,3-13C]AM by ip injection (47 mg/kg). Rats excreted 62 ± 12% of the dose in the 0 to 24-h urine. The percentage of excreted dose varied 1.5-fold among the four rats (range, 53 to 79%). The GSH-AM derived metabolites (1,1') accounted for approximately two-thirds of the excreted dose (69% of total excreted metabolites) following ip administration (Table 3Go). GA (metabolite 4; 7%) and GSH-GA-derived metabolites (metabolites 2,2' and 3,3'; 24%) accounted for the remainder.


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TABLE 3 Effect of Exposure Route on Urinary Metabolites in the Rat
 
On oral administration of acrylamide, data similar to those generated by ip administration were found. The GSH-AM derived metabolites (1,1') accounted for approximately two-thirds of the excreted dose (71% of total excreted metabolites) following ip administration (Table 3Go). GA (metabolite 4; 7%), GSH-GA-derived metabolites (metabolites 2,2' and 3,3'; 20%), and GA hydrolysis (metabolite 5, 1.7%) accounted for the remainder.

Following dermal application of 138 mg/kg [1,2,3- 13C]AM, rats excreted 1.5% (range, 0.43 to 2.8%) of the applied dose in the 0 to 24-h urine. Metabolites 1–4 were quantitated for the two rats with the highest percentage of the dermal dose excreted in urine (1.6 and 2.8%). Only metabolite 1,1' and GA could be detected and quantitated for the two additional rats. For the two rats in which quantitative values were obtained for metabolites 1–4, the AM-GSH-derived metabolites accounted for 46 to 58% of the total excreted metabolites (Table 3Go). For these two rats, GA accounted for 14 to 20% of the total excreted metabolites, and the GSH-GA-derived metabolites accounted for 28 to 34% of the total excreted metabolites.

A significant portion of the metabolites detected in urine from rats exposed to [13C]AM-vapor were derived from AM-GSH (1,1', 64% of the excreted metabolites, Table 3Go). Metabolites derived from GA-GSH (2,2', 3,3') accounted for 36% of the excreted metabolite, while GA was not detected in rats exposed via inhalation. In rats, the extent of oxidation via glycidamide (metabolites 2, 3, 4, and 5) was slightly higher on inhalation exposure when compared with po or ip administration. In contrast to rats, mice exposed to AM vapor had a similar percentage of metabolites attributed to GA (31%) and GSH-AM (27%), while GSH-GA accounted for 42% of the excreted metabolites (Table 4Go). In mice, approximately two-thirds of the urinary metabolites arise from oxidation of AM to GA.


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TABLE 4 Effect of Exposure Route on Urinary Metabolites in the Mouse
 
Hemoglobin Adducts of AM and GA
For quantitation of AAVal and GAVal, the AAVal and GAVal PTH derivatives were analyzed by LC-MS/MS in the negative ion mode with a turboionspray interface. The major ion formed was the parent ion (M-H-), and the major daughter ions resulted from loss of the AM or GA side chain. This provided the capability to distinguish among the adduct ions derived from AM, from [1,2,3-13C]AM, and from the internal standard labeled with valine-13C5, since the loss of the AM and GA side chains results in three distinct reactions that can be monitored to detect each form of the adduct (Fennell et al., 2003Go). For globin samples from rats and mice administered [1,2,3-13C]AM or administered [1,2,3-13C]AM in combination with [2,3-14C] AM, analyses were conducted for the adducts formed by both the 13C-enriched and natural abundance forms of AA and GA. Three separate chromatograms were obtained for two forms of the analyte and the internal standard. A similar set of chromatograms was obtained for GAVal in the same animal. For quantitation, the two peaks for the isomers of GAVal-PTH were integrated together.

The dermal administration of 150 mg/kg [13C]AM resulted in 13C-AAVal adduct levels that were approximately 10-fold lower than those observed following ip administration of 50 mg/kg [13C]AM in male rats (Table 5Go). 13C-GAVal levels were also lower (approximately 4-fold) on dermal administration compared with the ip administration. Adjusting for the difference in dose administered and comparing AAVal would suggest that approximately 3.6% of the administered dose is absorbed on dermal application of [13C]AM.


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TABLE 5 AM-Derived (AA-Val) and GA-Derived (GA-Val) Hemoglobin Adducts in Rats and Mice following Exposure to AM
 
A second analysis of dermal administration was conducted using a mixture of natural abundance AM, and [14C]AM. In this study, the levels of AAVal detected in globin were approximately 5-fold higher than observed in the study using the [13C]AM. (Table 5Go). Likewise the GAVal adduct levels were also approximately 4.5-fold higher than in the study using [13C]AM. Comparing the AAVal levels observed in the second dermal application with the ip administration suggests that approximately 16.5% of the applied dose was taken up. This calculation does not account for any differences in the conversion of acrylamide to glycidamide with dose route.

On inhalation exposure of rats and mice (Table 5Go), the amount of 13C-AAVal was similar in rats and mice and increased slightly between collection of blood immediately following exposure and at 24 h following exposure. The levels of 13C-GAVal also increased between the two time points. The amount of 13C-GAVal observed in the mouse was 3.6- and 3.8-fold that of the amount observed in the rat at the 0 and 24 h time points, respectively.

In Table 6Go, the amount of AAVal and GAVal formed under the various exposure scenarios was normalized to the amount of AM administered. For comparison, the data recently reported for acrylamide administered by gavage is included. Compared with gavage administration, ip administration produced lower AAVal but higher GAVal levels. With dermal administration, the amount of AAVal and GAVal calculated using the administered doses were lower than the other routes of exposure. However, when recalculated for the dose of AM that was recovered in excreta, carcass, and tissues (representing the amount of AM absorbed), the amounts of AAVal formed approached that found with po and ip administration, and the amount of GAVal formed with dermal administration was highest. With inhalation exposure in the rat, the amount of AAVal formed normalized to the dose taken up was lower than that formed with ip and gavage administration, but higher than that formed with dermal exposure. GAVal formed in the rat was similar to that formed with dermal and oral administration. In the mouse, which had the highest levels of AAVal and GAVal (Table 5Go), correction for the amount of AM taken in resulted in a considerably lower AAVal per mmol AM administered that found with the rat with inhalation, ip or po administration. This reflects a higher intake of acrylamide per kg body weight in the mouse, and indicates a more rapid metabolism of AM in the mouse. The amount of GAVal normalized per mmol of AM/kg body weight was similar between the rat and mouse.


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TABLE 6 Comparison of AAVal and GAVal per Unit of Administered Dose of AM in Rats and Mice
 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Occupational exposure to AM via dermal contact or inhalation exposure may potentially occur during its use as an intermediate in the production of polymers, or during the use of AM-containing grouting materials. In recent years, regulatory concerns about AM have risen due to the observation of adverse effects symptomatic of AM poisoning in tunnel workers using an AM-containing grouting agent (Hagmar et al., 2001Go). An important component in building an accurate regulatory framework for the industrial and commercial use of AM is the development of relevant toxicokinetic data for use in constructing risk assessment models. Toxicokinetic data are needed following multiple routes of administration because human exposure to AM can occur via dermal contact, inhalation exposure, or po administration (Dearfield et al., 1995Go; IARC, 1994Go). In general, tools for extrapolation between routes of exposure are not well defined, necessitating toxicokinetic data for each case scenario. In the present study, the metabolism and Hb adducts of AM were evaluated in rats following dermal application, ip injection, or inhalation exposure. AM metabolism and Hb adducts were also determined for mice exposed to AM vapor.

The extent of AM metabolism in rats on administration by gavage was similar in this study to our previous observations (Sumner et al., 1992Go). In this current study, rats administered [1,2,3-13C]AM by ip injection (47 mg/kg; 126 µmol) or gavage (59 mg/kg; 168 µmol) metabolized AM to a similar extent, with approximately 50–60% of the dose excreted in urine within 24 h. The relative metabolism via direct conjugation of GSH with AM vs. oxidation of AM to GA was similar for the ip and gavage administrations (Table 3Go). The majority of metabolism occurred via AM-GSH (67–71%) while a smaller portion was attributed to GA (6–7%), or GA-GSH (24–25%). In a previously reported study (Fennell et al., 2003Go), significantly higher levels of AAVal (20847 ± 1674 fmol/mg globin) were observed following oral administration of 50 mg AM/kg in rats compared with those reported in this study for ip administration (12900 ± 652 fmol/mg). GAVal levels were similar between ip (9031 ± 464 fmol/mg) and gavage (7876 ± 757 fmol/mg) administration. The ratio of GAVal:AAVal differed substantially between po (0.38 ± 0.07) and ip (0.71 ± 0.03) administration. Thus, while the overall extent of metabolism of AM to GA assessed from urinary metabolites is similar between ip and po administration, the internal dose of acrylamide is higher following oral administration. Normalizing AAVal and GAVal for the dose administered (Table 6Go) showed the higher amount of AAVal following oral administration. However, ip administration produced higher GAVal levels.

Following inhalation exposure to 2.9 ppm AM-vapor, rats had a smaller portion (31%) of the total recovered radioactivity (19 µmol) excreted in urine compared with gavage or ip administration. However, the percentage of urinary metabolites derived from GSH-AM (64%) was nearly identical to the other routes of administration. GA was not detected in urine of rats exposed to AM via inhalation, and the level of GA-GSH (37%) was slightly increased compared with ip and gavage exposures. When normalized for the dose administered (ip and oral) or the dose taken up (inhalation), the amount of AAVal formed was slightly lower on inhalation exposure in the rat. However, the amount of GAVal formed was highest on inhalation exposure.

Only 52% of the metabolites excreted in urine were derived from GSH-AM following dermal exposure to [13C]AM, with the remainder derived from GA (17%) or GSH-GA (21%). The GAVal to AAVal ratio was higher following dermal exposure (1.7) compared with ip injection (0.71) or inhalation (0.82 at 0 h; 1.1 at 24 h).

Dermal exposure of rats to 138 mg/kg [1,2,3-13C]AM (~11,000 µg/cm2 in distilled water; 382 µmol; same dosing solution as used for ip studies), resulted in only 1.5% of the applied dose excreted in urine, while dermal exposure to 168 mg/kg [2,3-14C]AM resulted in 8% of the applied dose excreted in urine. Hb adducts following dermal exposure to [13C]AM were ~5-fold lower than adducts measured following dermal exposure to [14C]AM. In vitro studies (Diembeck et al., 1998Go, cited with permission from TEGEWA) with pig skin indicated that low doses (1.3 µg/cm2) of AM (prepared in ethyl acetate) in creams and lotions had a 30% absorption, while higher doses (~2000 µg/cm2) of AM (in ethyl acetate) had a reduced absorption (~20%). The authors suggested that the lower absorbed dose following the 2000 µg/cm2 application of AM was attributed to a saturation in the penetration of AM. Absorption of 20–30% in vitro has been reported for low doses (<0.1 µg/cm2) of AM in polyacrylamide gels (Marty, 1998Go). Ramsey et al. reported (1984Go, cited with permission from SNF SA) that a 2 or 50 mg/kg application of [14C]AM (in 1% Triton-X450) to rats resulted in a 25% dermal absorption of the material. Thus, our results using [2,3-14C]AM are similar to those obtained from other laboratories using [14C]AM. Differences in the dermal penetration between our two studies using either [13C]AM or [14C]AM have not been clarified. Because the dosing solution used for the ip study (which produced results consistent with the literature) was the same as that used for the 13C-dermal study, it is unlikely that the in-life portion of the study was flawed. It is possible that trace impurities in either the 13C- or 14C-AM could either hinder or enhance, respectively, absorption.

The survival of rats and mice following a 6-h exposure to 5.6 ppm AM indicates that the LC50 for inhalation exposure is greater than 16 µg/l. Rats and mice metabolized AM differently following a 2.9-ppm exposure to AM-vapor. AM metabolism in mice occurred largely following P450 oxidation (73% of total excreted metabolites), while this pathway was smaller (36%) for rats. A much smaller portion of metabolism occurred via AM-GSH (27%) in mice, compared with rats (64%). GA (31%) was detected in urine from mice exposed to AM vapor, but was not detected in urine from exposed rats. GA-GSH accounted for 42 or 37% of the excreted metabolites in mice or rats, respectively. AAVal levels were similar between rats and mice exposed via inhalation. However, the ratio of GAVal to AAVal was significantly higher in mice compared with rats, consistent with a higher P450 oxidation in mice. When normalized by body weight, the total recovered radioactivity in mice (8 µmol) was nearly 2.8-fold higher than recovered for rats (18 µmol). Likewise, the ratio of the mouse:rat total adduct level (AAVal + GAVal) was 2.4:1, indicating a greater relative uptake of AM in mice compared with rats. The normalized AAVal data (Table 6Go) on inhalation exposure indicate a much higher extent of formation of AAVal in the rat per unit dose compared with the mouse. However, the amount of GAVal in the mouse normalized per unit dose was comparable to that observed in the rat (Table 6Go). The higher minute ventilation (Arms and Travis, 1988Go) for mice (1.5 ml/min/g) compared with rats (0.7 ml/min/g), and the greater capacity of mice for P450 oxidation of AM may account for the higher relative uptake of AM and lower AAVal levels in mice on inhalation exposure.

While rats had a similar flux through pathways following gavage administration or inhalation exposure, mice had significant differences between these two routes of exposure. Mouse metabolism via AM-GSH was greater following a 50 mg/kg (17 µmol) gavage administration (41%, Sumner et al., 1992Go) than inhalation exposure (27%), while this pathway was significantly higher in rats following either exposure route (64–67%). Mice had higher GA levels (31%) and GSH-GA (42%) following inhalation exposure compared with gavage administration (17%, 33%, respectively). These data may indicate route differences in the metabolism of AM in mice, or possible dose-dependent differences due to the lower uptake of AM following inhalation exposure (2.9 ppm, 8 µmol) compared with gavage administration (50 mg/kg, 17 µmol). However, rats had a similar flux through the AM-GSH pathway following a 50 mg/kg ip injection or a 2.9-ppm inhalation exposure.

A pharmacokinetic model based on hemoglobin adducts of AM and GA in the rat (Calleman et al., 1992Go) predicted that 22% of AM is converted to GA following a 50 mg/kg ip dose. This prediction is consistent with results from the present study (ip) and previous research (gavage, Sumner et al., 1992Go) that indicated a 30% conversion of AM to GA in rats. Models have not been developed for AM following inhalation or dermal routes of exposure for rats, nor have any models been developed for mice. Data on the metabolism, distribution, and pharmacokinetics of AM following inhalation or dermal exposures in rats and mice are needed for the development of models aimed at extrapolating human health risks.


    ACKNOWLEDGMENTS
 
The CIIT animal care staff are acknowledged for technical support. This study was supported by the Polyelectrolyte Producers Group and the Acrylamide Monomer Producers Association (Dr. Marvin Freidman, primary scientific contact). We thank Dr. Barbara Kuyper for editorial review.


    NOTES
 
1 Present address: Paradigm Genetics, Inc., P.O. Box 14528, Research Triangle Park, NC 27709-2548. Back

2 Present address: RTI International, P.O. Box 12194, Research Triangle Park, NC 27709-2194. Back

3 Present address: GlaxoSmithKline, Research Triangle Park, NC 27709. Back

4 To whom correspondence should be addressed at present address: RTI International, P.O. Box 12194, 3040 Cornwallis Road, Research Triangle Park, NC 27709-2914. Fax: (910) 541-6499. E-mail: fennell{at}rti.org. Back


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