ToxSci Advance Access originally published online on July 26, 2006
Toxicological Sciences 2006 93(2):256-267; doi:10.1093/toxsci/kfl069
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Kinetics of Elimination of Urinary Metabolites of Acrylamide in Humans
* RTI International, Research Triangle Park, North Carolina 27709; and UMDNJ, Newark, New Jersey 07103
1 To whom correspondence should be addressed at RTI International, P.O. Box 12190, 3040 Cornwallis Road, Research Triangle Park, NC 27709. Fax: (919) 541-6499. E-mail: fennell{at}rti.org.
Received February 23, 2006; accepted July 24, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Acrylamide (AM), used in the manufacture of polyacrylamide and grouting agents, is produced during the cooking of foods. Workplace exposure to AM can occur through the dermal and inhalation routes. The objective of this study was to define the kinetics of elimination of AM and its metabolites following oral and dermal administration. This is the second part of a study in which metabolites and hemoglobin adducts of AM were determined in people (Fennell et al., 2005, Toxicol. Sci. 85, 447–459). (1,2,3-13C3)AM was administered in an aqueous solution orally (single dose of 0.5, 1.0, or 3.0 mg/kg) or dermally (three daily doses of 3.0 mg/kg) to sterile male volunteers. Urine samples were collected at 0–2, 2–4, 4–8, 8–16, and 16–24 h following administration orally, or at 0–2, 2–4, 4–8, 8–16, and 16–24 h following each of three daily dermal doses. 13C3-AM and its metabolites in urine, 13C3-glycidamide, 13C3-N-acetyl-S-(3-amino-3-oxopropyl)cysteine and its S-oxide, and 13C3-N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine, were quantitated using liquid chromatography-tandem mass spectrometry. The recovered urinary metabolites accounted for 45.6, 49.9, and 39.9% of a 0.5, 1.0, and 3.0 mg/kg oral dose (0–24 h), respectively, and for 4.5% of the dose after 3 mg/kg was administered daily for 3 days dermally (0–4 days). These results indicate that after oral administration AM is rapidly absorbed and eliminated. The half-life estimated for elimination of AM in urine was 3.1–3.5 h. After dermal administration, AM uptake is slow. This study indicated that skin provides a barrier that slows the absorption of AM, and results in limited systemic availability following dermal exposure to AM.
Key Words: acrylamide; glycidamide; NACP sulfoxide; urinary metabolites.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Acrylamide (AM) is used in the manufacture of polyacrylamide, and is used in grouting agents. Polyacrylamide is used in the manufacture of paper, for sludge dewatering, as a flocculant in water treatment, and is used in the manufacture of consumer products. Concern over human exposure has been primarily with workplace exposure to grouting agents, with dermal and inhalation exposure the most likely routes (European Union, 2002
).
The discovery of AM in fried and baked foods (Rosen and Hellenas, 2002; Tareke et al., 2000, 2002) has aroused considerable concern about the potential health effects of the widespread exposure to low levels of AM in foods (WHO, 2002).
AM is carcinogenic in rats (Friedman et al., 1995; Johnson et al., 1986), producing mammary gland adenocarcinomas and fibroadenomas in female rats, thyroid follicular cell adenomas and adenocarcinomas in male and female rats, and mesotheliomas of the testicular tunic in male rats (Friedman et al., 1995). AM is neurotoxic, inducing a characteristic peripheral neurotoxicity in animals and man (Spencer and Schaumburg, 1974a,b, 1975). This toxicity is manifested as a distal to proximal loss of nerve function and dying back of cells. AM is a germ cell mutagen (Dearfield et al., 1995), and affects rodent reproduction, producing reduced litter size. At elevated AM doses other reproductive effects are seen, likely as a consequence of dominant lethal mutations at low doses and neurotoxicity at higher doses (Tyl and Friedman, 2003).
The role of metabolism in the mode of action of AM has been the subject of some research. AM is reactive, and can react directly with proteins. AM reacts rapidly with the sulfhydryl group of glutathione (GSH), and the N-acetylcysteine conjugate of AM is a major metabolite of AM in urine (Fig. 1). AM also undergoes oxidation to glycidamide (GA), a reactive epoxide (Calleman et al., 1990; Sumner et al., 1992). This metabolite is then further metabolized via hydrolysis or GSH conjugation or can react with proteins, including hemoglobin, and with DNA (Gamboa da Costa et al., 2003; Segerbäck et al., 1995). The oxidation of AM to GA is catalyzed primarily by cytochrome P450-2E1 (Sumner et al., 1999). One of the primary issues in risk assessment for AM is whether AM or GA is the active form in generating the various toxic effects, or whether both are capable of generating toxic effects. GA is genotoxic, reacts with DNA, and is generally assumed to be the agent of concern in carcinogenesis (Paulsson et al., 2001). GA is thought to mediate the heritable mutational events caused by AM exposure (Favor and Shelby, 2005; Ghanayem et al., 2005a,b). The role of AM and GA in the generation of neurotoxic effects is less clear, with one study indicating that GA is active (Abou-Donia et al., 1993), whereas others concluded that AM is the active agent (Barber et al., 2001; Costa et al., 1992, 1995). The extent of metabolism of AM to GA is thus one of the factors to be considered in conducting a human health risk assessment.
|
Dermal exposure to AM has been evaluated in a number of studies, examining the penetration of AM in vitro in pig skin (Diembeck et al., 1998
), and in vivo in rodents to evaluate uptake and metabolism (Carlson and Weaver, 1985; Ramsey et al., 1984; Sumner et al., 2003), or effects (Adler et al., 2004; Bull et al., 1984a,b; Gutierrez-Espeleta et al., 1992). In vivo studies in rats have suggested that AM absorption when administered dermally is considerably less than the applied dose, with estimates of 1.5 and 8% of the applied dose excreted in urine from two studies. From hemoglobin adduct measurements in the same studies, 3.6 and 16.5% uptake was estimated (Sumner et al., 2003).
The primary objectives of this study were to evaluate the conversion of AM to GA in people exposed to AM, and to evaluate the extent of uptake following dermal administration. This was conducted by administering 13C-substituted AM to volunteers orally or dermally, and measuring urinary metabolites or hemoglobin adducts derived from the GA pathway and comparing them with those derived from AM directly. We have previously reported the initial results of this study (Fennell et al., 2005), in which metabolites were analyzed in a single pooled sample of urine for each individual administered AM at the highest dose, using 13C nuclear magnetic resonance (NMR) spectroscopy. This approach enabled quantitation of the total urinary metabolites excreted in the collection period, but the limited sensitivity of the method precluded analysis of lower doses administered orally, and dermal administration. Here we report further analysis, in which we have developed a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for analysis of AM and its urinary metabolites, and applied it to urine samples collected at 0–2, 2–4, 4–8, 8–16, and 16–24 h after administration of a single oral dose of AM (0.5, 1.0, or 3.0 mg/kg) and after one, two, and three doses of AM administered dermally (3.0 mg/kg). The objective of this study was to define the kinetics of elimination of AM and its metabolites following oral and dermal administration.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Details of the administration of AM to human volunteers have been reported previously and are reviewed in brief below (Fennell et al., 2005
).
Institutional Review Board Approval
This study was conducted in accordance with the Code of Federal Regulations (CFR) governing Protection of Human Subjects (21 CFR 50), Institutional Review Board (IRB) (21 CFR 56), retention of data (21 CFR 312) as applicable and consistent with the Declaration of Helsinki. The administration of 13C AM to the study subjects was conducted at Covance Clinical Laboratories. IRB approval of the protocol and the consent form was obtained at Covance Clinical Research Unit (CRU). Written informed consent was obtained from all study participants prior to study participation. IRB approval was also obtained at RTI International, where the analysis of the samples was conducted. The study participants were compensated for time spent in the Covance CRU. The stipends were reviewed by the IRB as part of the study protocol, and those approved for this study were within the general stipend range approved by the IRB for other studies conducted at Covance CRU.
Chemicals
(1,2,3-13C3) AM (CLM-813, lot number 11085, 99% labeled) was obtained from Cambridge Isotopes Limited. Identity and purity were confirmed by 1H and 13C NMR spectroscopy. (Note that the molecular weight of the (1,2,3-13C3) AM is 74, vs. 71.08 for natural-abundance AM.)
AM Exposure
Twenty-four volunteers participated in this study. They were all male Caucasians (with the exception of one Native American) weighing between 71 and 101 kg and between 26 and 68 years of age. All volunteers were aspermic and had not used tobacco products for the past 6 months. They passed a drug screen and had not taken prescription drugs or caffeinated products over the previous 3 days. They had not consumed alcohol-containing beverages or medications within 7 days of study entry, and for the duration of the study. Each experimental group consisted of six individuals of which one was a placebo. There were two phases to this study: an oral phase and a dermal phase.
In the oral phase, three groups of six people were administered 0.5, 1.0, or 3.0 mg/kg 13C3 AM. Individuals were presented with test substance at approximately 9:00 A.M. to initiate the study. Urine was collected at 0–2, 2–4, 4–8, 8–16, and 16–24 h.
In the dermal phase, a 50% (wt/vol) solution of 13C3 AM was applied directly on the skin to a clean, dry, marked off, 24 cm2 (3 cm x 8 cm) area on the volar forearm. After applying the appropriate amount of material, the liquid was evaporated to dryness using a commercial hair dryer and covered with a sterile gauze pad. After drying the AM solution, the tape which had been used to demark the area of application was removed and placed in a vial containing 20 ml of water. The water (dermal dam solution) was analyzed for AM by high-performance liquid chromatography (HPLC). The site of application was covered with gauze for 24 h at which time the gauze was removed and the area was washed with 1000 ml of water. The recovered wash water was analyzed by HPLC for AM. Dermal applications alternated between left and right arms, starting with the subject's dominant arm. Blood was collected immediately prior to compound administration and 24, 48, 72, and 96 h later (immediately prior to administration of the second and third doses, after gauze removal and prior to leaving the clinic). Hormone blood samples were drawn immediately prior to compound administration, after 24 h and on day 5 when the volunteers left the clinic.
Each exposure group contained six volunteers. Of the six volunteers in each group, five received the designated amount of AM, and one received no AM. AM was applied to the skin for 24 h on one forearm, and a blood sample was collected at 24 h following the first administration. This was repeated on the following 2 days, with AM applied on alternating arms, for a total of three dermal doses of AM at 24 h intervals. A total of five blood samples was collected from each volunteer administered AM dermally, on day 1 (prior to the first dose), day 2, day 3, day 4, and day 5. The sample obtained on day 5 was at 24 h following removal of the occlusion at the site of application.
Urine samples were collected at intervals of 0–2, 2–4, 4–8, 8–16, and 16–24 h following administration of AM. The volume of urine in each sample was recorded, and sample aliquots were transferred to sample vials for storage.
Urinary Metabolites of AM
Standards.
Unlabeled standards were prepared for GA, cysteine-S-propionamide (CP), N-acetyl cysteine-S-propionamide (NACP), N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine (GAMA2), N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine (GAMA3), and NACP sulfoxide (Fig. 1). 13C3-substituted standards were prepared for CP, NACP, and NACP sulfoxide.
NMR and mass spectral analysis of standards.
NMR spectra were recorded using a Bruker AMX 300-MHz spectrometer, or a Varian Inova 500-MHz spectrometer. Mass spectra of standards were obtained on an Applied Biosystems (Foster City, CA) API 3000 mass spectrometer equipped with an Agilent 1100 LC and a Turboionspray interface.
Synthesis of GA. GA was synthesized by H2O2 oxidation of acrylonitrile (Payne and Williams, 1961), as described previously (Fennell et al., 2003).
Synthesis of CP. CP was synthesized by reaction of cysteine with AM in a solution of triethylamine in water, essentially as described by Calleman et al. (1990). The identity of the product was confirmed by 1H NMR (not shown) and by mass spectrometry. 13C3-substituted CP was synthesized the same way except with 13C3-AM.
Mass spectrum, Turboionspray, direct infusion, positive ion, m/z 193.1, 176.1, 146.9, 130.0, 103.9, 76.0, 71.9, 55.1.
Mass spectrum 13C3 substituted, m/z 195.9, 179.1, 150.1, 133.1, 106.9, 75.2, 72.0, 58.2.
Synthesis of NACP. NACP was synthesized by reacting approximately 2 mmol each of N-acetylcysteine, AM, and triethylamine in 3.6 ml of water. The reaction was incubated for 6 days at 35°C. The product was lyophilized, and purified by reverse-phase HPLC using a Waters Atlantis dC18 column (250 mm x 4.6 mm, 5 µm). NACP was eluted with 0.1% formic acid using the same HPLC system. Substituted NACP was synthesized the same way with 13C3-AM.
1H NMR (300 MHz) 
2.08 (3H, s, CH3), 2.58 (2H, t, CH2-CONH2), 2.86 (2H, t, S-CH2-CH2), 2.98 (1H, dd, cys ß), 3.15 (1H, dd, cys ß'), and 4.50 ppm (1H, dd, cys 
).
Mass spectrum, Turboionspray, direct infusion, positive ion, m/z 234.9, 216.9, 192.9, 189.3, 147.2, 130.2, 104.1.
Mass spectrum 13C3 substituted, m/z 237.9, 220.3, 195.9, 191.9, 150.2, 133.1, 107.1.
Synthesis of GAMA. GA (1.2 mmol) was reacted with N-acetylcyteine (1.3 mmol), triethylamine (1.4 mmol), and 2 ml of water. The reaction was incubated at 35°C for 5 days. The product was purified by reverse-phase HPLC using a Waters Atlantis dC18 column (250 mm x 4.6mm, 5 µm) with guard column, and was eluted with 0.1% formic acid using the same HPLC system. Two peaks were collected, peak A and peak B.
Peak A: 1H NMR spectrum (300 MHz) 2.04 (3H, s, CH3), 3.0 (1H, 2 dd, cyst ß), 3.15 (1H, 2 dd, cys ß'), 3.55 (1H, t, S-CH-CONH2), 3.80 (2H, m, S-CH-CH2-OH), and 4.50 ppm (1H, dd, cys ).
13C NMR (75 MHz) 21.9 (CH3), 33.1 and 33.3 (cys ß), 50.2 and 50.5 (S-CH), 61.79 and 61.83 (CH2-OH), 56.3 (cys ) signals assigned to carbonyl carbons were detected but not assigned at 175 ppm.
The 1H and the 13C NMR spectra were consistent with the formation of a pair of diastereomers, based on the multiplicity of the signals for the cysteine ß-protons, the carbon signals for the cysteine ß carbon, and the methylene and methine carbons derived from AM. Based on the NMR data, this metabolite is assigned the structure GAMA2 (corresponding to metabolite 3 described in Sumner et al., 1992).
Peak A: Mass spectrum, Turboionspray, direct infusion, positive ion, m/z 251.5 (M+H+) daughter ions at 233.2, 209.1, 191.4, 174.4, 162.1, 146.1, 130.1, 128.1.
Peak B: 1H NMR spectrum (300 MHz) 2.04 (3H, s, CH3), 2.9 (1H, m, S-CH-CH2-OH), 3.0 (2H, m, cyst ß, and S-CH-CH'2-OH), 3.15 (1H, m, cys ß'), 4.38 ppm (1H, dd, cys ), and 4.59 (1H, dt, S-CH-CONH2).
13C NMR (75 MHz): 21.8 (CH3), 33.7 and 33.8(cys ß), 36.1 and 36.3 (S-CH2-CHOH), 53.2 and 53.3 (cys ), 70.57 and 70.59 (CHOH-CONH2), 174.3 (CH3-CO), 174.72 and 174.76 (CO2H), and 178.0 ppm (CONH2).
The 1H and the 13C NMR spectra were consistent with the formation of a pair of diastereomers based on the multiplicity of the signals for the cysteine ß-protons, the carbon signals for the cysteine ß carbon, and the methylene and methine carbons derived from AM. Based on the NMR data, this metabolite is assigned the structure GAMA3 (corresponding to metabolite 2 described in Sumner et al., 1992).
Peak B: Mass spectrum, Turboionspray, direct infusion, positive ion, m/z 251.5 (M+H+) daughter ions at 232.9, 209.1, 205.1, 192.1, 174.4, 163.1, 145.9, 130.1, 120.1.
Synthesis of NACP sulfoxide. NACP (17.2 mg), water (344 µl), and H2O2 (3.4 µl) were mixed and placed at 4°C overnight. The reaction was purified by reverse-phase HPLC using the same column and HPLC system as NACP. The substituted compound was synthesized similarly.
1H NMR (500 MHz): 1.90, 1.96 (3H, s, CH3), 2.63 (2H, q, CH2-CONH2), 2.98 (1H, m, S-CH2a-CH2), 3.11 (2H, m, S-CH2b-CH2, and cys ß), 3.25 and 3.50 (0.5 H each, dd, cys ß'), and 4.55 ppm (1 H, 2 dd, cys ).
13C NMR (75 MHz): 21.95 and 21.97 (CH3), 27.88 and 27.95 (S-CH2-CH2), 46.89 and 46.94 (CH2-CONH2), 49.01 and 49.52 (cys ß), 53.49 and 53.59 (cys ), 174.0 (CONH), 174.25 and 174.46 (CO2H), and 175.75 and 175.78 (CONH2).
Mass spectrum, Turboionspray, direct infusion, positive ion, m/z 250.9 (M+H+), 235.3 (M+H+ – 16), 216.3, 173.4, 162.1, 117.6, 101.8.
Mass spectrum 13C3 substituted, m/z 254 (M+H+), 237.1, 236.1, 218.1, 194.1, 180.1, 176.1, 162.1, 129.9, 125.1, 120.1, 101.1.
Sample Preparation
Urine samples were thawed and 65 µl was taken for analysis. Internal standard solutions were prepared with individual standard at concentrations of 20 µg/ml, except for GA and NACP which were used at a concentration of 80 µg/ml. Each unlabeled internal standard solution (5 µl of each) was added to each sample and standard curve sample to give a final volume of 100 µl. Each sample was vortexed and centrifuged at 14,000 rpm for 2 min, and 10 µl was transferred to a microfuge tube. Ninety microliters of water was added and the tubes were vortexed and centrifuged at 14,000 rpm for 2 min. The sample was transferred to a low volume insert for LC-MS/MS analysis.
LC-MS/MS Analysis of Urinary Metabolites
An API 3000 LC-MS/MS system with Agilent 1100 binary pump, degasser, and autosampler was used for analysis. The column used was a Waters (Milford, MA) Atlantis dC18 (250 mm x 4.6 mm, 5 µm), with guard column. The flow rate was 1 ml/min and was split with 50% going to the MS/MS. AM and its metabolites were eluted with 0.1% formic acid in water. After 25 min, the column was washed with 95% acetonitrile, and 5% of 0.1% formic acid in water for 5 min. A 5-min gradient was run to return the column to its starting conditions, and reequilibration was conducted for 10 min.
The sample injection volume was 10 µl. Elution was monitored by multiple reaction monitoring (MRM) in the positive ion mode. The ion source was Turboionspray. The nebulizer gas was set at 10 (arbitrary units), the curtain gas at 8, the collision gas was set at 4, the ionization voltage was set at 4500 V, and the source temperature was 500°C.
Analysis was conducted by selected reaction monitoring (SRM) of selected precursor 
product ion transitions for each analyte and unlabeled standard:
- AM, m/z 75 58, 72 55
- GA, m/z 91
74, 88 71
- CP, m/z 196
107, 193 104
- NACP, m/z 238
196, 235 193
- GAMA2, m/z 254
162, 251 162
- GAMA3, m/z 254
149, 251 146
- NACP sulfoxide, m/z 254
162, 251 162.
- GA, m/z 91
Standard curves were used to calculate the concentration of each metabolite in urine when available (AM, CP, NACP, and NACP sulfoxide). Other metabolites were quantitated by the ratio of analyte to the internal standard added. The range of concentrations used for standard curves provided a linear response, and is indicated below:
- AM, range 0.1–10 µg/ml, r2 = 0.998
- CP, range 0.1–5 µg/ml, r2 = 0.995
- NACP, range 0.5–200 µg/ml, r2 = 0.983
- NACP sulfoxide, range 0.1–100 µg/ml, r2 = 0.993.
- CP, range 0.1–5 µg/ml, r2 = 0.995
For each metabolite, a limit of quantitation (LOQ, signal:noise of 10:1) and a limit of detection (LOD, signal:noise of 3:1) corresponded to
- AM, LOQ = 0.002 µg/ml, LOD = 0.001 µg/ml
- CP, LOQ = 0.0018 µg/ml, LOD = 0.0008 µg/ml
- NACP, LOQ = 0.25 µg/ml, LOD = 0.08 µg/ml
- NACP Sulfoxide, LOQ = 0.01 µg/ml, LOD = 0.005 µg/ml
- GA, LOQ = 0.06 µg/ml, LOD = 0.03 µg/ml
- GAMA3, LOQ = 0.008 µg/ml, LOD = 0.005 µg/ml
- GAMA2, LOQ = 0.008 µg/ml, LOD = 0.005 µg/ml.
- CP, LOQ = 0.0018 µg/ml, LOD = 0.0008 µg/ml
Analyses were conduced in duplicate, with triplicate standard curves, and with quality control standards incorporated.
Pharmacokinetic Analysis of Urinary AM
The kinetics of AM elimination in urine following oral administration of AM were analyzed using WinNonlin version 4.0.1 from Pharsight (Cary, NC). Noncompartmental analysis using Model 210 for extravascular input and urine data was used to calculate the elimination rate constant and half-life.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
With one exception, the major metabolites of AM have been previously reported, and characterized (Calleman et al., 1990
; Dixit et al., 1982; Sumner et al., 1992). We have recently reported the detection of a sulfoxide metabolite of NACP in human urine by 13C NMR spectroscopy (Fennell et al., 2005). Here we have verified that the chemical shifts reported in urine for the metabolite detected by 13C NMR (6b, 27.60 ppm; 6a, 46.47 ppm; and 6c, 175.4 ppm) correspond to the signals assigned to the AM-derived carbon atoms in a synthetic standard (27.88 and 27.95 for S-CH2-CH2; 46.89 and 46.94 for CH2-CONH2; 175.75 and 175.78 for CONH2). The standard consisted of a pair of diastereomers, which could be separated by HPLC.
Method Development for Analysis of Urinary Metabolites
A sensitive method for quantitation of urinary metabolites of AM was developed using LC-MS/MS. The metabolites investigated were chosen based on our previous study, which involved the identification and quantitation of urinary metabolites of (1,2,3-13C3) AM by NMR spectroscopy (Fennell et al., 2005). Unlabeled standards were prepared for GA, glyceramide, NACP, CP, NACP sulfoxide, GAMA3, and GAMA2. Isotope-substituted standards were prepared for NACP, CP, and NACP sulfoxide, using (1,2,3-13C3) AM as starting material. Standard curves were prepared for the isotope-substituted standards, and natural-abundance standards. However, since the administered material was (1,2,3-13C3) AM, the analyte standard was the isotope-substituted form, and the internal standard was the natural-abundance form. Concentrations were calculated using the ratio of analyte:internal standard from the standard curve. For metabolites for which there was no isotope-substituted standard available (derived from GA), the ratio of analyte:internal standard was used directly to calculate the concentration of analyte. Urine samples were prepared directly by addition of internal standards, dilution, and centrifugation.
For chromatographic separation of AM and its metabolites, isocratic elution with 0.1% formic acid in water was used. This enabled the early elution of ion-suppressing materials from the column, and permitted the retention of AM and GA and the resolution of the diastereomers of NACP sulfoxide, GAMA2, and GAMA3. A representative chromatogram of a urine sample from a subject administered 13C3 AM orally in Figure 2 shows the peaks derived from the isotope-substituted metabolites of 13C3-AM. LC-MS/MS in the positive ion mode enabled the sensitive detection of each of the metabolites of interest. The chromatograms associated with the natural-abundance standards are available in Supplementary Data. AM and GA could be readily identified and quantitated: 1,2,3-13C3 AM appears as a single peak in Figure 2A, with a retention time of 7.1 min, and 1,2,3-13C3 GA (Fig. 2B) had a retention time of 5.4 min. However, glyceramide was poorly retained on a variety of columns evaluated, and could not be quantitated because of the interference from ion-suppressing materials in urine that coeluted with glyceramide. Thus, the data in this paper are presented without the quantitation of glyceramide.
|
Resolution of the metabolites derived from GSH conjugation also required isocratic elution with 0.1% formic acid in water. 13C3-CP (MRM 196
107) was eluted at a retention time of 4.1 min (Fig. 2C). Peaks were observed at other retention times for the precursor product ion transition of m/z 196 107, but were clearly resolved from 13C3-CP. The major metabolite which gave this transition, as well as m/z 238 196 was 13C3-NACP at a retention time of 23.9 min (Fig. 2D). Since GAMA2, GAMA3, and NACP sulfoxide are isomeric, and do not give unique fragments on LC-MS, chromatographic resolution of these materials was required. For each of the two isomers of the N-acetylcysteine conjugates of GA, two diastereomers could be resolved. 13C3-NACP sulfoxide is also present as a pair of diastereomers, and is isomeric with the N-acetylcysteine conjugates of 13C3-GA. Thus, a total of six peaks appear in common in Figure 2E and 2F. The peaks for each isomer/diastereomer could be clearly resolved chromatographically. The major GA conjugate (GAMA3), which corresponds to metabolite 2 (Sumner et al., 1992), was present as two peaks at 11.9 and 12.3 min (Fig. 2E). GAMA2, which corresponds to metabolite 3 (Sumner et al., 1992), was present as two peaks at 8.3 and 8.8 min (Fig. 2F). 13C3-NACP sulfoxide was present as a pair of diastereomers at 6.8 and 7.3 min (Fig. 2F).
The analytical approach used in this study in which unlabeled internal standard was added to each sample has the potential to have slight errors due to the potential contribution of metabolites of exogenous unlabeled dietary AM. However, given the relatively high concentration of standards added, and the low levels of AM metabolites that are likely in the normal background urine, it is unlikely that this will be a significant source of error. Boettcher et al. (2005) have recently reported median levels of NACP of 60 µg/l (range of 3–338 µg/l). The levels of NACP used as internal standard in this study were 60 µg/ml. Boettcher et al. (2005) have also recently reported median levels of GAMA3 of 8 µg/l (range of < LOD – 45 µg/l). The level of GAMA3 used as internal standard in this study was 20 µg/ml. Therefore, we expect that the background of metabolites from dietary AM would not interfere significantly with the measurements made in this study.
Oral Administration
In urine samples following oral intake of AM, the following were measured: AM, GA, NACP and its S-oxide, and two isomers of the GA mercapturic acids.
Unchanged 13C3-AM accounted for approximately 9–10% of the urinary metabolites measured (Table 1). The elimination of AM in urine occurred at early time points, with the majority excreted by 4 h. 13C3-GA accounted for approximately 1% of the urinary metabolites. Low levels of the AM cysteine conjugate, 13C3-CP, were detected accounting for 0.4–0.6% of the urinary metabolites. As described previously for a limited number of samples (Fennell et al., 2005), the major metabolite of AM was NACP. This metabolite accounted for 68–69% of the excreted metabolites measured in all three dose groups. The sulfoxide of NACP, which had been previously reported in human urine (Fennell et al., 2005), accounted for 17–19% of the urinary metabolites. The two mercapturic acid conjugates of GA together accounted for less than 2% of the metabolites in urine. The predominant form of this conjugate was the 3-substituted form (GAMA3). The recovery of AM and its metabolites in urine following oral administration expressed as a percentage of the administered dose was 39.9 ± 9.9% at the high dose, to 49.9 ± 6.3% at the mid dose, and 45.6 ± 8.5% at the low dose.
|
Pharmacokinetic analysis of the excretion of AM was conducted, using a noncompartmental model. The rate of elimination and the elimination half-life estimated at each exposure level were similar, with the mean half-life ranging from 3.1 to 3.5 h (Table 2). In contrast to AM, which was eliminated primarily in the 0–2 and 2–4 h urine samples, the maximal amounts of the metabolites were eliminated in the 8–16 h samples. Analysis of the formation and elimination of metabolites was beyond the scope of a simple noncompartmental analysis.
|
Dermal Administration
In the majority of samples of urine from volunteers receiving AM dermally, GA was not detected (Table 3). AM was not detected in the 0–2 h sample from the first day of administration. The only metabolite that appeared in the 0–2 h sample was 13C3-CP, which was detected at low levels in urine samples at first time point from three of the five exposed individuals. The concentration detected was above the LOD, but below LOQ, and has been included in Table 3 for completeness. In the 2–4 h sample collected on the first day, both AM and CP were detectable. NACP and its sulfoxide were not detectable until the 4–8 h urine samples from day 1. The GA mercapturic acids were not detectable until the 8–16 h samples. NACP was the largest metabolite excreted in urine, accounting for 69.7% of the urinary metabolites. NACP sulfoxide was the next most abundant metabolite, accounting for 22.4% of the urinary metabolites. Approximately 4% was recovered as AM, and approximately 3% was derived from GA (GA and its mercapturic acid metabolites, glyceramide was not measured). The rate of excretion (µmol/h, Fig. 3) of NACP was maximal on day 3, following the third administration of AM dermally. AM and its metabolites were excreted on day 4, indicating that up to 2 days following the last dermal dose, AM was being absorbed and excreted. However, the rate of excretion of AM dropped from a peak of 0.27 µmol/h reached on day 3 to 0.016 µmol/h at the end of day 4, suggesting that absorption and elimination of AM were nearing completion.
|
|
In our previous study, we had reported two measures of dose: the applied dose, which consisted of the AM applied to the skin; the absorbed dose, which consisted of the AM applied to the skin—the AM washed off the skin at the end of the exposure. The recovered urinary metabolites accounted for 4.5 ± 0.8% of the applied dose (Table 4), and 12.4 ± 3.1 of the absorbed dose (Table 5) (Fennell et al., 2005
).
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
This report provides an extension of the previous publication (Fennell et al., 2005
) of urinary metabolites reported using 13C NMR for analysis of urinary metabolites of (1,2,3-13C3) AM. The use of LC-MS/MS for the analysis has enabled quantitation of metabolites in each urine sample collected, which in turn has provided pharmacokinetic data, and a comparison of data obtained at three dose levels following oral administration, and following dermal administration. In comparing the data reported here and those reported previously, it should be noted that a single composite sample for each subject was prepared for analysis by NMR spectroscopy (reported previously), whereas individual samples collected over a number of time intervals were prepared for LC-MS/MS analysis. AM was detected but not quantitated by NMR spectroscopy, and glyceramide was not quantitated by LC-MS. Previously, we reported that 34 ± 5.7% of the dose was recovered following oral administration of AM (Fennell et al., 2005). In this study, the recovered metabolites accounted for 39.9 ± 9.9% of the administered dose. Metabolites that were present at low concentration, and could not be quantitated by NMR, namely metabolites 2 and 3, the GA mercapturic acids, were quantitated by LC-MS.
Of considerable interest is whether the metabolism of AM changes at low doses. The oxidation of AM to GA is catalyzed by CYP2E1 (Sumner et al., 1999), and is saturable (Calleman et al., 1993). Studies by Calleman have suggested that the percentage of AM metabolized via GA increases dramatically with decreasing dose. While this is the case in making comparisons over the range of 0.5–100 mg/kg in rodents, in the range employed in this human study, there did not appear to be any significant differences between the doses studied with respect to the percentage of metabolites present as GA, and its mercapturic acid conjugates.
AM is excreted directly in urine. Previous studies in which NMR spectroscopy was used to quantitate the metabolites of 13C3 AM had detected but not quantitated AM. This was because of the long relaxation time of AM. In the current study, we have quantitated AM excreted with time, and conducted a pharmacokinetic analysis of AM. The half-life or elimination rate constant of AM has been estimated previously in a number of publications. Calleman (1996) estimated that the elimination rate constant of AM in humans was 0.23 h–1 by extrapolation from data obtained in rats. Our measurements ranged from 0.21 to 0.26 h–1, or half-lives of 3.1–3.5 h. Sorgel et al. (2002) reported half-lives of AM in two subjects of 2.2 and 7 h, following ingestion of AM-containing food. Fuhr et al. (2006) recently reported a half-life of 2.4 ± 0.4 h for AM following ingestion of AM-containing food.
AM is rapidly taken up and excreted in urine, following oral administration. This contrasts with dermal exposure, in which there is a lag period of several hours before AM appears in the urine. This is consistent with the time required for AM to cross the stratum corneum and penetrate the dermis. One of the motivating factors for measuring the AM metabolites in urine was that the systemic availability derived from hemoglobin adduct measurements was approximately 6.6% of that estimated if the dermally administered dose was entirely absorbed (Fennell et al., 2005). Since the extent of adduct formation depends on area under the curve in blood, which in turn depends on the dose taken up, and the rate of elimination, verification of the extent of uptake and elimination of urinary metabolites was necessary to substantiate the measurement of availability from hemoglobin adduct measurements. In this study, approximately 4.5% of the dermally administered dose was recovered as urinary metabolites. When a more refined dose metric of dermally absorbed dose was compared with hemoglobin adduct measurements, an estimate of 17.0% availability was obtained (Fennell et al., 2005). Approximately 12.35% of the absorbed dose was recovered as urinary metabolites measured by LC-MS. Thus, it appears that the absorption of AM estimated from urinary metabolites and from hemoglobin adducts is consistent. The values obtained in this study indicate that the estimates that have been used in risk assessments for AM as a result of dermal exposure, e.g., 75% (European Union, 2002), appear to be overly conservative.
A limitation of the approach taken in analyzing the urinary metabolites by LC-MS/MS was that we did not succeed in quantitating glyceramide in urine. Since glyceramide accounts for a considerable fraction of the metabolism of GA following oral administration, it is not possible to estimate the fractions of AM administered metabolized by direct GSH conjugation and that via oxidation to GA. Previously, after analysis of urinary metabolites by NMR spectroscopy we reported that glyceramide accounted for approximately 11% of the urinary metabolites following oral administration of 3 mg/kg AM, and for approximately 80% of the GA-derived metabolites detected (Fennell et al., 2005). Development of a sensitive method for quantitation of glyceramide in urine is a priority for assessing metabolism via GA in humans.
In this study, a lag between the first application of 1,2,3-13C3 AM dermally and the appearance of AM and its metabolites in the urine was apparent. This lag is consistent with the time required for AM to penetrate the stratum corneum and to penetrate the dermis. The percutaneous absorption of chemicals is frequently described as a steady-state diffusion process following Fick's Law. However, the steady state condition is achieved some time after the initial contact with the skin. The lag time required to reach steady-state conditions can be estimated based on the path length of chemical diffusion, commonly the thickness of the stratum corneum, and diffusion coefficient in the membrane. A lag time of 0.24 h has been estimated based on the physicochemical properties of AM (Hoang, 1992). Organic chemicals can form a reservoir in the stratum corneum, resulting in slow release to the systemic circulation (Hadgraft, 1979). The time course of appearance of AM and its metabolites in urine is consistent with a slow release of AM from the skin. The application of AM was terminated at 72 h, and urine was collected for a further 24 h following the end of the exposure. While AM metabolites were still being eliminated at the end of day 4, the rate of excretion of AM dropped from a peak of 0.27 µmol/h reached on day 3 to 0.016 µmol/h at the end of day 4. The rate of formation of hemoglobin adducts during the same period resulted in a decline, estimated from the mean difference in AAVal and GAVal between each day and the preceding day (Fig. 4). The rate of formation was maximal on days 3 and 4, following the second and third dermal applications of AM. On day 5, after removal of the last application of AM, the increment formed has fallen considerably compared with the days during which an application took place.
|
Interestingly, S-(3-amino-3-oxopropyl)cysteine was detected earliest in urine following dermal exposure, in the absence of AM, suggesting that glutathione conjugation may occur in skin. Complete first pass metabolism in the skin at low levels of dermal exposure is a possibility. Skin contains GSH, is known to express GSH transferases, and has the capability to metabolize substrates such as dinitrochlorobenzene in vitro (Jewell et al., 2000
). AM applied dermally is known to decrease the concentration of GSH in skin (Mukhtar et al., 1981).
It is clear from this study that the extent of metabolism of AM via GA in humans is considerably lower than that reported following oral administration in rats (41% at 3 mg/kg) and mice (59% at 50 mg/kg) (Fennell et al., 2005; Sumner et al., 1992). A number of recent reports have appeared which describe the measurement of the mercapturic acid metabolites of AM and GA in the urine of smokers and nonsmokers, in the urine of an individual who had ingested 1 mg deuterated AM, and in the urine of volunteers who ingested food containing 1 mg AM (summarized in Table 6). In the study reported by Bjellaas et al. (2005), GAMA3 and NACP were detected and quantitated in urine from one smoker and five nonsmokers. Urine samples were collected at intervals over 3 days, during which the volunteers fasted between breakfast on day 1 and breakfast on day 2. The ratio of GAMA3:NACP was reported as 0.46 for nonsmokers in this study. It is possible that the period of fasting may alter CYP2E1 activity, although conflicting effects of fasting on CYP2E1 activity have been reported in rats (O'Shea et al., 1994) and in humans (Wan et al., 2006). Boettcher et al. (2005) reported that the median concentration of the mercapturic acids of AM (NACP) and GA (GAMA, assigned as N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine) was 60 and 8 µg/l, respectively, in urine. The ratio of GAMA:NACP for individual samples was reported to be between 0 and 0.5 (Boettcher et al., 2006), with a median ratio of 0.16. Following ingestion of 1 mg d3-AM by a single volunteer, urine samples were collected for 2 days, and the excretion of d3-NACP and d3-GAMA in urine was measured by LC-MS/MS (Boettcher et al., 2006). A total of 56.2% of the dose administered was recovered as urinary metabolites, with 4.6% as d3-GAMA, and 51.7% as d3-NACP. In the first 22 h following administration, 2.7% of the administered dose was excreted as d3-GAMA. This corresponds to the observation of 0.5–0.7% of the dose following oral administration, eliminated as GAMA3 in this study. It appears that the extent of metabolism as a result of conversion to GA and its mercapturic acids in this study was lower than that reported by others. Following administration of AM (1 mg) in food to volunteers, AM, NACP, and GAMA3 excreted in urine accounted for 4, 50, and 6% of the dose (Fuhr et al., 2006) (GAMA:NACP of 0.118). Urban et al. (2006) described a GAMA:NACP ratio of 0.23 for nonsmokers and 0.15 for nonsmokers. In contrast with the previous studies, we report a ratio of GAMA:NACP of 0.024–0.026. While the differences could result from differences between administered AM, versus the low doses of AM in food, or from the sampling time following ingestion of AM, the method used for analysis may be a significant factor. The method reported here includes analysis of NACP sulfoxide in addition to GAMA2 and GAMA3. These metabolites give the same molecular ion, and have many fragments in common. Chromatographic separation is thus required for quantitation. For the natural-abundance forms of these three metabolites with analysis by LC-MS/MS in the negative ion mode, we have observed (Fig. 5) that peaks for NACP sulfoxide, GAMA2, and GAMA3 are visible when monitoring m/z 249 120 (conditions similar to those used by Boettcher and Angerer, 2005 to quantitate GAMA3). Boettcher et al. (2005) measured NACP and GAMA3 in urine in smokers and nonsmokers using negative ion LC-MS/MS monitoring m/z 249 120, with an LC system resolved a single peak for GAMA3 at 9.25 min and NACP at 11.86 min. It is possible that NACP sulfoxide is not resolved chromatographically from GAMA3 under those conditions, and thus could be quantitated inadvertently as GAMA3. This same method has been used in a number of publications (Boettcher et al., 2006; Fuhr et al., 2006).
|
|
While NACP and GAMA have been used as urinary metabolites for monitoring exposure to AM, and to characterize metabolism to GA, it is apparent from this study that NACP sulfoxide should be included in the measurement of metabolites in urine. Since hydrolysis appears to account for the majority of the GA metabolites in urine (Fennell et al., 2005
), development of a method for analysis of glyceramide in urine should be a priority. |
SUPPLEMENTARY DATA |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Supplementary data are available online at http://toxsci.oxfordjournals.org/.
|
NOTES |
|---|
The authors certify that all research involving human subjects was done under full compliance with all government policies and the Helsinki Declaration.
|
ACKNOWLEDGMENTS |
|---|
This study was funded by SNF SA. However, SNF does not have control over the resulting publication. We would like to acknowledge Dr William Bridson and Ms Rebecca Spicer of the Covance CRU, Inc. for the conduct of the clinical portion of the study (reported previously in Fennell et al., 2005).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
|---|
Abou-Donia, M. B., Ibrahim, S. M., Corcoran, J. J., Lack, L., Friedman, M. A., and Lapadula, D. M. (1993). Neurotoxicity of glycidamide, an acrylamide metabolite, following intraperitoneal injections in rats. J. Toxicol. Environ. Health 39, 447–464.[Web of Science][Medline]
Adler, I. D., Gonda, H., Hrabe de Angelis, M., Jentsch, I., Otten, I. S., and Speicher, M. R. (2004). Heritable translocations induced by dermal exposure of male mice to acrylamide. Cytogenet. Genome Res. 104, 271–276.[CrossRef][Web of Science][Medline]
Barber, D. S., Hunt, J. R., Ehrich, M. F., Lehning, E. J., and LoPachin, R. M. (2001). Metabolism, toxicokinetics and hemoglobin adduct formation in rats following subacute and subchronic acrylamide dosing. Neurotoxicology 22, 341–353.[CrossRef][Web of Science][Medline]
Bjellaas, T., Janak, K., Lundanes, E., Kronberg, L., and Becher, G. (2005). Determination and quantification of urinary metabolites after dietary exposure to acrylamide. Xenobiotica 35, 1003–1018.[CrossRef][Web of Science][Medline]
Boettcher, M. I., and Angerer, J. (2005). Determination of the major mercapturic acids of acrylamide and glycidamide in human urine by LC-ESI-MS/MS. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 824, 283–294.[Web of Science][Medline]
Boettcher, M. I., Bolt, H. M., Drexler, H., and Angerer, J. (2006). Excretion of mercapturic acids of acrylamide and glycidamide in human urine after single oral administration of deuterium-labelled acrylamide. Arch. Toxicol. 80, 55–61.[CrossRef][Web of Science][Medline]
Boettcher, M. I., Schettgen, T., Kutting, B., Pischetsrieder, M., and Angerer, J. (2005). Mercapturic acids of acrylamide and glycidamide as biomarkers of the internal exposure to acrylamide in the general population. Mutat. Res. 580, 167–176.[Web of Science][Medline]
Bull, R. J., Robinson, M., Laurie, R. D., Stoner, G. D., Greisiger, E., Meier, J. R., and Stober, J. (1984a). Carcinogenic effects of acrylamide in Sencar and A/J mice. Cancer Res. 44, 107–111.
Bull, R. J., Robinson, M., and Stober, J. A. (1984b). Carcinogenic activity of acrylamide in the skin and lung of Swiss-ICR mice. Cancer Lett. 24, 209–212.[CrossRef][Web of Science][Medline]
Calleman, C. J. (1996). The metabolism and pharmacokinetics of acrylamide: Implications for mechanisms of toxicity and human risk estimation. Drug Metab. Rev. 28, 527–590.[Web of Science][Medline]
Calleman, C. J., Bergmark, E., and Costa, L. G. (1990). Acrylamide is metabolized to glycidamide in the rat: Evidence from hemoglobin adduct formation. Chem. Res. Toxicol. 3, 406–412.[CrossRef][Web of Science][Medline]
Calleman, C. J., Bergmark, E., Stern, L. G., and Costa, L. G. (1993). A nonlinear dosimetric model for hemoglobin adduct formation by the neurotoxic agent acrylamide and its genotoxic metabolite glycidamide. Environ. Health Perspect. 99, 221–223.[Web of Science][Medline]
Carlson, G. P., and Weaver, P. M. (1985). Distribution and binding of [14C]acrylamide to macromolecules in SENCAR and BALB/c mice following oral and topical administration. Toxicol. Appl. Pharmacol. 79, 307–313.[CrossRef][Web of Science][Medline]
Costa, L. G., Deng, H., Calleman, C. J., and Bergmark, E. (1995). Evaluation of the neurotoxicity of glycidamide, an epoxide metabolite of acrylamide: Behavioral, neurochemical and morphological studies. Toxicology 98, 151–161.[CrossRef][Web of Science][Medline]
Costa, L. G., Deng, H., Gregotti, C., Manzo, L., Faustman, E. M., Bergmark, E., and Calleman, C. J. (1992). Comparative studies on the neuro- and reproductive toxicity of acrylamide and its epoxide metabolite glycidamide in the rat. Neurotoxicology 13, 219–224.[Web of Science][Medline]
Dearfield, K. L., Douglas, G. R., Ehling, U. H., Moore, M. M., Sega, G. A., and Brusick, D. J. (1995). Acrylamide: A review of its genotoxicity and an assessment of heritable genetic risk. Mutat. Res. 330, 71–99.[CrossRef][Web of Science][Medline]
Diembeck, W., Dusing, H.-J., and Akhiani, M. (1998). Dermal Absorption and Penetration of Acrylamide ([C14]-Acrylamide as Tracer) in Different Cosmetic Formulations and Polyacrylamide-Solution after Topical Application to Excised Pig Skin. Beiersdorf.
Dixit, R., Seth, P. K., and Mukjtar, H. (1982). Metabolism of acrylamide into urinary mercapturic acid and cysteine conjugates in rats. Drug Metab. Dispos. 10, 196–197.[Web of Science][Medline]
European Union. (2002). European Union Risk Assessment Report—Acrylamide, 210 pp. Office for Official Publications of the European Communities, Luxembourg.
Favor, J., and Shelby, M. D. (2005). Transmitted mutational events induced in mouse germ cells following acrylamide or glycidamide exposure. Mutat. Res. 580, 21–30.[Web of Science][Medline]
Fennell, T. R., Sumner, S. C., Snyder, R. W., Burgess, J., Spicer, R., Bridson, W. E., and Friedman, M. A. (2005). Metabolism and hemoglobin adduct formation of acrylamide in humans. Toxicol. Sci. 85, 447–459.
Friedman, M. A., Dulak, L. H., and Stedham, M. A. (1995). A lifetime oncogenicity study in rats with acrylamide. Fundam. Appl. Toxicol. 27, 95–105.[CrossRef][Web of Science][Medline]
Fuhr, U., Boettcher, M. I., Kinzig-Schippers, M., Weyer, A., Jetter, A., Lazar, A., Taubert, D., Tomalik-Scharte, D., Pournara, P., Jakob, V., et al. (2006). Toxicokinetics of acrylamide in humans after ingestion of a defined dose in a test meal to improve risk assessment for acrylamide carcinogenicity. Cancer Epidemiol. Biomarkers Prev. 15, 266–271.
Gamboa da Costa, G., Churchwell, M. I., Hamilton, L. P., Von Tungeln, L. S., Beland, F. A., Marques, M. M., and Doerge, D. R. (2003). DNA adduct formation from acrylamide via conversion to glycidamide in adult and neonatal mice. Chem. Res. Toxicol. 16, 1328–1337.[CrossRef][Web of Science][Medline]
Ghanayem, B. I., Witt, K. L., El-Hadri, L., Hoffler, U., Kissling, G. E., Shelby, M. D., and Bishop, J. B. (2005a). Comparison of germ cell mutagenicity in male CYP2E1-null and wild-type mice treated with acrylamide: Evidence supporting a glycidamide-mediated effect. Biol. Reprod. 72, 157–163.
Ghanayem, B. I., Witt, K. L., Kissling, G. E., Tice, R. R., and Recio, L. (2005b). Absence of acrylamide-induced genotoxicity in CYP2E1-null mice: Evidence consistent with a glycidamide-mediated effect. Mutat. Res. 578, 284–297.[Web of Science][Medline]
Gutierrez-Espeleta, G. A., Hughes, L. A., Piegorsch, W. W., Shelby, M. D., and Generoso, W. M. (1992). Acrylamide: Dermal exposure produces genetic damage in male mouse germ cells. Fundam. Appl. Toxicol. 18, 189–192.[CrossRef][Web of Science][Medline]
Hadgraft, J. (1979). Epidermal reservoir—Theoretical approach. Int. J. Pharm. 2, 265–274.
Hoang, K. T. (1992). Dermal Exposure Assessment: Principles and Applications (U.S. Environmental Protection Agency, Ed.). Office of Health and Environmental Assessment, Washington, DC.
Jewell, C., Heylings, J., Clowes, H. M., and Williams, F. M. (2000). Percutaneous absorption and metabolism of dinitrochlorobenzene in vitro. Arch. Toxicol. 74, 356–365.[CrossRef][Web of Science][Medline]
Johnson, K. A., Gorzinski, S. J., Bodner, K. M., Campbell, R. A., Wolf, C. H., Friedman, M. A., and Mast, R. W. (1986). Chronic toxicity and oncogenicity study on acrylamide incorporated in the drinking water of Fischer 344 rats. Toxicol. Appl. Pharmacol. 85, 154–168.[CrossRef][Web of Science][Medline]
Mukhtar, H., Dixit, R., and Seth, P. K. (1981). Reduction in cutaneous and hepatic glutathione contents, glutathione S-transferase and aryl hydrocarbon hydroxylase activities following topical application of acrylamide to mouse. Toxicol. Lett. 9, 153–156.[CrossRef][Web of Science][Medline]
O'Shea, D., Davis, S. N., Kim, R. B., and Wilkinson, G. R. (1994). Effect of fasting and obesity in humans on the 6-hydroxylation of chlorzoxazone: A putative probe of CYP2E1 activity. Clin. Pharmacol. Ther. 56, 359–367.[Web of Science][Medline]
Paulsson, B., Granath, F., Grawe, J., Ehrenberg, L., and Tornqvist, M. (2001). The multiplicative model for cancer risk assessment: Applicability to acrylamide. Carcinogenesis 22, 817–819.
Ramsey, J. C., Young, J. D., and Gorzinski, S. J. (1984). Acrylamide: Toxicodynamics in Rats. Health and Environmental Sciences, Toxicology Research Laboratory, Dow Chemical U.S.A., Midland, MI.
Rosen, J., and Hellenas, K. E. (2002). Analysis of acrylamide in cooked foods by liquid chromatography tandem mass spectrometry. Analyst 127, 880–882.[Medline]
Segerbäck, D., Calleman, C. J., Schroeder, J. L., Costa, L. G., and Faustman, E. M. (1995). Formation of N-7-(2-carbamoyl-2-hydroxyethyl)guanine in DNA of the mouse and the rat following intraperitoneal administration of [14C]acrylamide. Carcinogenesis 16, 1161–1165.
Sorgel, F., Weissenbacher, R., Kinzig-Schippers, M., Hofmann, A., Illauer, M., Skott, A., and Landersdorfer, C. (2002). Acrylamide: Increased concentrations in homemade food and first evidence of its variable absorption from food, variable metabolism and placental and breast milk transfer in humans. Chemotherapy 48, 267–274.[CrossRef][Web of Science][Medline]
Spencer, P. S., and Schaumburg, H. H. (1974a). A review of acrylamide neurotoxicity. Part I. Properties, uses and human exposure. Can. J. Neurol. Sci. 1, 143–150.[Medline]
Spencer, P. S., and Schaumburg, H. H. (1974b). A review of acrylamide neurotoxicity. Part II. Experimental animal neurotoxicity and pathologic mechanisms. Can. J. Neurol. Sci. 1, 152–169.[Medline]
Spencer, P. S., and Schaumburg, H. H. (1975). Nervous system degeneration produced by acrylamide monomer. Environ. Health Perspect. 11, 129–133.[Medline]
Sumner, S. C., Fennell, T. R., Moore, T. A., Chanas, B., Gonzalez, F., and Ghanayem, B. I. (1999). Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice. Chem. Res. Toxicol. 12, 1110–1116.[CrossRef][Web of Science][Medline]
Sumner, S. C., MacNeela, J. P., and Fennell, T. R. (1992). Characterization and quantitation of urinary metabolites of [1,2,3-13C]acrylamide in rats and mice using 13C nuclear magnetic resonance spectroscopy. Chem. Res. Toxicol. 5, 81–89.[CrossRef][Web of Science][Medline]
Sumner, S. C., Williams, C. C., Snyder, R. W., Krol, W. L., Asgharian, B., and Fennell, T. R. (2003). Acrylamide: A comparison of metabolism and hemoglobin adducts in rodents following dermal, intraperitoneal, oral, or inhalation exposure. Toxicol. Sci. 75, 260–270.
Tareke, E., Rydberg, P., Karlsson, P., Eriksson, S., and Törnqvist, M. (2000). Acrylamide: A cooking carcinogen? Chem. Res. Toxicol. 13, 517–522.[CrossRef][Web of Science][Medline]
Tareke, E., Rydberg, P., Karlsson, P., Eriksson, S., and Tornqvist, M. (2002). Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J. Agric. Food Chem. 50, 4998–5006.[CrossRef][Web of Science][Medline]
Tyl, R. W., and Friedman, M. A. (2003). Effects of acrylamide on rodent reproductive performance. Reprod. Toxicol. 17, 1–13.[CrossRef][Web of Science][Medline]
Urban, M., Kavvadias, D., Riedel, K., Scherer, G., and Tricker, A. R. (2006). Urinary mercapturic acids and a hemoglobin adduct for the dosimetry of acrylamide exposure in smokers and nonsmokers. Inhal. Toxicol. 18, 831–839.[CrossRef][Web of Science][Medline]
Wan, J., Ernstgard, L., Song, B. J., and Shoaf, S. E. (2006). Chlorzoxazone metabolism is increased in fasted Sprague-Dawley rats. J. Pharm. Pharmacol. 58, 51–61.[Web of Science][Medline]
WHO. (2002). Joint FAO/WHO Consultation on Health Implication of Acrylamide in Food, 33 pp. World Health Organization, Geneva, Switzerland.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. I. Ghanayem, R. Bai, and L. T. Burka Effect of Dose Volume on the Toxicokinetics of Acrylamide and Its Metabolites and 2-Deoxy-D-glucose Drug Metab. Dispos., February 1, 2009; 37(2): 259 - 263. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 O. Doroshyenko, U. Fuhr, D. Kunz, D. Frank, M. Kinzig, A. Jetter, Y. Reith, A. Lazar, D. Taubert, J. Kirchheiner, et al. In vivo Role of Cytochrome P450 2E1 and Glutathione-S-Transferase Activity for Acrylamide Toxicokinetics in Humans Cancer Epidemiol. Biomarkers Prev., February 1, 2009; 18(2): 433 - 443. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Bjellaas, P. T. Olesen, H. Frandsen, M. Haugen, L. H. Stolen, J. E. Paulsen, J. Alexander, E. Lundanes, and G. Becher Comparison of Estimated Dietary Intake of Acrylamide with Hemoglobin Adducts of Acrylamide and Glycidamide Toxicol. Sci., July 1, 2007; 98(1): 110 - 117. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||