United States Environmental Protection Agency Environmental Monitoring Systems Laboratory P.O. Box 93478 Las Vegas NV 89193-3478 EPA 600/R-93/226 November 1993 Preissue copy Research and Development &EPA Determination of Hemoglobin Ad ducts Following Acrylamide Exposure 9312-0181ead ------- DETERMINATION OF HEMOGLOBIN ADDUCTS FOLLOWING ACRYLAMIDE EXPOSURE Lucio G. Costa, Ph.D., Principal Investigator Carl J. Calleman, Ph.D., Co-Principal Investigator Department of Environmental Health, SC-34 University of Washington Seattle, WA 98195 Cooperative Agreement CR-816768-01-0 Project Officer: Charles H. Nauman, Ph.D., M.P.H. Exposure Assessment Research Division Environmental Monitoring Systems Laboratory Las Vegas, Nevada 89193 ENVIRONMENTAL MONITORING SYSTEMS LABORATORY OFFICE OF RESEARCH AND DEVELOPMENT US ENVIRONMENTAL PROTECTION AGENCY LAS VEGAS, NEVADA 89193 ------- NOTICE The information contained in this document has resulted from research funded by the United States Environmental Protection Agency through Cooperative Agreement CR-816768-01-0 to the University of Washington, Seattle, Washington. It has been subjected to the Agency's peer and administrative review, and it has been approved for publication as an EPA Project Report. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. 11 ------- ABSTRACT The present project was undertaken to develop new methodologies for biological monitoring of exposure to the toxicant acrylamide in laboratory animals as well as humans. Methods were developed to measure the adducts of acrylamide and its epoxide metabolite glycidamide to cysteine in rat hemoglobin and to valine in human hemoglobin by means of gas chromatography/mass spectrometry. Studies in rats indicated that both acrylamide and glycidamide adducts are formed following acute or chronic exposure to acrylamide, while only the glycidamide adducts are formed after exposure to glycidamide. Both adducts, in addition to acrylonitrile adducts, were measured in a group of Chinese workers exposed to acrylamide during its synthesis and polymerization. Significant signs of neurotoxicity were also found in this population. Additional studies in rats indicated that acrylamide, but not glycidamide, is the proximate neurotoxicant while glycidamide may be responsible for the male reproductive and genotoxic/carcinogenic effects of acrylamide. These studies suggest that these novel biomarkers to assess exposure to acrylamide are useful to assess potential health hazards (including possible cancer risks) due to exposure to acrylamide in occupationally exposed workers as well as in the general population. This report was submitted in fulfillment of Cooperative Agreement CR-816768-01-0 by the University of Washington under the sponsorship of the U.S. Environmental Protection Agency. The report covers a project period from September 1990 to September 1992, with a no-cost extension until March 1993 to permit analysis of the extensive field data collected in the People's Republic of China. Work was completed as of March 1993. Funding for the research was provided by the Office of Research and Development (ORD), Environmental Monitoring Systems Laboratory, Las Vegas, Exposure Biomarkers Research Program, under the Toxic Substances Budget Sub-Activity (L104). Partial support for the field trip to the People's Republic of China was provided by the ORD Health Effects Research Laboratory (HERL), Research Triangle Park, NC, under the ORD/HERL program in Research to Improve Health Risk Assessments (REHRA). 111 ------- CONTENTS NOTICE ii ABSTRACT iii FIGURES vi TABLES vii ABBREVIATIONS ix ACKNOWLEDGMENTS x INTRODUCTION 1 Acrylamide 1 Determination of hemoglobin adducts for exposure assessment 2 AIMS 2 METHODS 4 Aiml: 4 Synthesis of deuterated analogues 4 Treatment of animals 5 Isolation of globin and analysis of hemoglobin adducts 5 Gas chromatography/mass spectrometry analysis 6 Alkylation of hemoglobin in vitro 6 Aim 2: 6 Synthesis of alkylated valines 6 NMR Spectroscopy and fast atom bombardment mass spectrometry (FAB-MS) 9 Analysis of Hb adducts in globin hydrolysates .. 9 Analysis of hemoglobin adducts with the modified Edman degradation method 9 Gas chromatography-mass spectrometry analysis 9 Alkylation of hemoglobin in vitro 10 Calibration 10 Field study in the People's Republic of China 11 Physical description of the factory 11 Air monitoring 11 Description of study populations 11 Determination of mercapturic acids in urine 13 Blood samples for hemoglobin adducts 13 Measurements of vibration thresholds 14 Electroneuromyography (ENMG) 14 Clinical and laboratory examinations 15 Questionnaire information 15 IV ------- Definition of neurotoxicity index 15 Statistical methods 15 Aim 3: 17 Animals and treatments 17 Neurotoxicity testing 17 Reproductive toxicity testing 17 RESULTS AND DISCUSSION 18 Aim 1: 18 Development of a GC/MS method for measuring Hb adducts of AA and GA 18 Acrylamide and glycidamide hemoglobin adducts following administration to rats 19 General discussion of Aim 1 27 Aim 2: 29 Development of methods for acrylamide and glycidamide Hb adducts in humans 29 Symptoms and signs in acrylamide exposed workers 30 Vibration thresholds 30 Eiectroneuromyographic measurements 34 The neurotoxicity index as predictor of clinical diagnosis of peripheral neuropathy 34 Hb adduct levels in humans exposed to acrylamide 34 Calculation of in vivo doses 44 Other biomarkers of exposure 44 Biomarkers of exposure as predictors of neurotoxicity 44 General discussion of Aim 2 .. 52 Hemoglobin adducts 52 Field study 54 Aim 3: 58 Comparative neurotoxicity of acrylamide and glycidamide 58 Behavioral tests 58 Morphological observations 58 Male reproductive toxicity of acrylamide and glycidamide 62 General discussion of Aim 3 • 62 REFERENCES 65 ------- FIGURES Figure 1 Reactions of AA and GA with cysteine residues in Hb 3 Figure 2 Reactions of AA, GA and AN with the N-terminal valine of Hb 8 Figure 3 Hb cysteine adducts of AA and GA in rats injected with 20 different single doses of AA Figure 4 Mass fragmentogram from a rat treated with 10 mg/kg AA 21 Figure 5 Hb - cysteine adducts of GA in rats injected with different 22 single doses of GA Figure 6 Conversion of AA to GA in rats injected with AA 23 Figure 7 Neurotoxicity Index in workers with and without clinical diagnosis 36 of peripheral neuropathy Figure 8 Mass fragmentogram of AA and GA valine adducts 39 Figure 9 Correlation between AA and GA Hb adducts in workers 40 exposed to AA Figure 10 Correlation between neurotoxicity (NIn) and levels of free 46 AA in plasma Figure 11 Correlation between neurotoxicity (NIn) and urinary .. 47 mercapturic acids levels Figure 12 Correlation between neurotoxicity (NIn) and AA Val adducts 48 Figure 13 Correlation between neurotoxicity (NIn) and AN Val adducts 49 Figure 14 Correlation between neurotoxicity (NIn) and lifetime in vivo 50 doses of AA Figure 15 Body weights of rats treated with AA or GA v 59 Figure 16 Rotarod test performance of rats exposed to AA or GA 60 Figure 17 Hindlimb splay test in rats exposed to AA or GA 61 VI ------- TABLES Table la - Air concentration of acrylamide (mean +. SD, mg/m5) 12 Table Ib - Air concentrations of acrylonitrile (mean ± SD, mg/m3) 12 Table 2 - Definition of Neurotoxicity Index 16 Table 3 - Hemoglobin adducts in rats treated with different dose 24 regimens of acrylamide Table 4 - Pharmacokinetic parameters of acrylamide and 26 glycidamide in the rat Table 5 - Second-order rate constants for the reaction of acrylamide 31 and glycidamide with amino acid residues in hemoglobin Table 6 - Percentage of acrylamide-exposed workers and controls 32 with symptoms of adverse health effects Table 7 - Comparison of vibration thresholds (VU ± SD) between 33 acrylamide workers and referents Table 8 - Comparison of motor (MCV) and sensory (SCV) nerve 35 conduction between the acrylamide and reference groups Table 9 - Hemoglobin adducts of acrylamide (AAVal), glycidamide 37 (GAVal) and acrylonitrile (ANVal) in workers Table 10 - Air concentrations of AA and AAVal Hb adduct levels 41 in workers in different locations Table 11 - Individual values for parameters predictive of adverse 42 health outcome in workers exposed to acrylamide Table 12 - Biomarkers according to job classification 45 Table 13 - Correlation coefficients and levels of statistical 51 significance for the relationships between different predictive parameters and the Neurotoxicity Index vn ------- Table 14 - Effects of acrylamide and glycidamide on male 63 reproductive organs in rats Table 15 - Effects of acrylamide and glycidamide on male 64 reproductive parameters in rats Vlll ------- ABBREVIATIONS AA Acrylamide AACys S-(2-carbamoylethyl)cysteine AAVal N-(2-carbamoylethyl)valine ANOVA Analysis of Variance ANVal N-(2-cyanoethyl)valine Bq Bequerel CEVal N-(2-carboxyethyl)valine CHEVal N-(2-caiboxy-2-hydroxyethyl)valine EI^o Effect Level, 50% El Electron impact EMG Electromyography ENMG Electroneuromyography eV Electronvolt FAB-MS Fast Atom Bombardment Mass Spectrometry GA Glycidamide GACys S-(2-carbamoyl-2-hydroxyethyl)cysteine GAVal N-(2-carbamoyl-2-hydroxyethyl)valine GC/MS Gas Chromatography/Mass Spectrometry Hb Hemoglobin HFB Heptafluorobutyryl HFBA Heptafluorobutanoic Anhydride HOEtVal Hydroxylethyl Valine HPLC High Pressure Liquid Chromatography LOEL Lowest Observed Effect Level MCV Motor Nerve Conduction Velocity MeOH Methanol NIn Neurotoxicity Index NMR Nuclear Magnetic Resonance NOEL No Observed Effect Level PCI Positive Chemical lonization PFPITC Pentafluorophenyl isothiocyanate PFPTH Pentafluorophenylthiohydantoin SEM Standard Error of the Mean SCV Sensory Nerve Conduction Velocity TLC Thin-Layer Chromatography TLV Threshold Limit Value TWA Time-Weighted Average VU Vibration Units IX ------- ACKNOWLEDGEMENTS This research project was carried out in the Department of Environmental Health at the University of Washington in Seattle. In addition to the Principal Investigator and Co-Principal Investigator, a major contributor was Dr. Emma Bergmark, who based her Ph.D. thesis on this work. Dr. David Kalman and Russell Dills were instrumental in facilitating the mass spectrometry work. Drs. F. He, H. Deng, Y. Wu, S. Zhang and Y. Wang from the Institute of Occupational Medicine at the Chinese Academy of Preventive Medicine in Beijing (PRC) and Drs. G. Tian and B. Xia from the Institute of Occupational Health of XinXiang City, Hunan Province, (PRC) were important collaborators for the field study in China. Dr. Charles Nauman, Project Officer, EPA, offered useful guidance in the development and realization of this project. Chris Sievanen provided secretarial assistance. ------- INTRODUCTION Acrylamide At room temperature acrylamide is a white crystalline solid with a high solubility in water. The high reactivity of its olefinic bond versus nucleophilic agents in Michael type additions is derived from its conjugation with an amide group. Industrially, acrylamide is produced by catalytic hydration of acrylonitrile and used mainly in the production of polymers such as polyacrylamide. Humans are potentially exposed to acrylamide in industrial processes, grouting operations, synthesis of chromatography gels and leakage of the monomer from polyacrylamide used in the purification of drinking water. According to a survey undertaken by NIOSH, some 10,000 workers in 27 occupations are potentially exposed to acrylamide, including about one thousand persons involved in the synthesis of polyacrylamides and another thousand licensed to perform grouting operations. The number of laboratory workers exposed to acrylamide in the preparation of chromatography gels has been estimated to be as high as 100,000-200,000. Since the early fifties the neurotoxic effects, involving both the central and the peripheral nervous system, have been the primary health concern of human exposure to acrylamide; close to 150 cases of human intoxication have been reported in the last 30 years (He et al. 1989). These reports have been paralleled by extensive studies in experimental animals, notably aiming at the elucidation of the mechanism of neurotoxic action of acrylamide (Miller and Spencer, 1985; Tilson, 1981). In more recent years, following the demonstration that acrylamide induces tumors in mice and rats, attention has been increasingly focused on the genotoxic and reproductive effects of the compound (Dearfield et al. 1988). Thus, acrylamide has been shown to be mutagenic in vitro in mouse lymphoma cells, and to give rise to heritable translocations and dominant lethal mutations in rodents. Unexpectedly for a compound producing these types of effects, acrylamide has consistently given a negative response in the Ames/'Salmonella assay both in the absence and in the presence of an S-9 fraction. Despite the fact that acrylamide has been shown to induce this wide range of adverse effects in mammalian species, few studies have investigated the role of metabolism in the toxicity of this compound and although reports have appeared indicating that acrylamide may undergo further metabolism, the structures of the intermediates generated have not been elucidated. From the point of view of quantitative risk assessment of human exposure this has represented a serious gap of knowledge, since the extrapolation of toxic effects in animals requires not only the identification of the agent(s) responsible for the induction of the effects, but, ideally, also information about the relationship between exposure dose and in vivo dose (defined as the time-integrated concentration of free electrophilic agent in vivo). In addition, since exposure to acrylamide may occur through several different routes, the total amount absorbed by a person may be very difficult to assess. ------- Determination of hemoglobin adducts for exposure assessment The structural elucidation and quantitative determination of hemoglobin (Hb) adducts can be used to obtain this type of information. For example, in response to long-standing speculation about the possible involvement of a cytochrome P-450-generated agent in the toxicity of acrylamide, we were recently able to demonstrate the formation in vitro and in vivo in rats of glycidamide, an epoxide formed by oxidation of the olefinic bond of acrylamide, through the identification of S-(2-carboxy-2-hydroxyethyl)cysteine (Fig. 1) in hydrolyzed Hb samples from acrylamide exposed rats (Calleman et al. 1990). Determination of Hb adducts as a way of assessing the relationship between environmental concentrations and in vivo dose, as well as for the dosimetry-based risk estimation of toxic effects, has been undertaken for several types of alkylating agents in humans. The advantages of such measurements are: - Hb adducts are stable during the 4-month life span of the human erythrocyte, meaning that in vivo doses are integrated over a long and well-defined period of time. - Large quantities of Hb are easily available from exposed animals and humans allowing for detection of low levels of adducts, e.g. by gas chromatography-mass spectrometry. - Differences between species and individuals with regard to rates of uptake, metabolic activation and deactivation of electrophiles, as well as induction status of enzymes, are reflected in the levels of Hb adducts formed in vivo, thus providing a basis for extrapolation between species and dosimetry-based risk estimation. - The relationship between dose in red blood cells and in target organs can be studied in experimental animals, and extrapolated to humans if due consideration is given to reaction kinetic and physicochemical parameters of the electrophile. A method for the determination of Hb adducts of the parent compound acrylamide has been developed by Bailey et al. (1986) based on the liberation of S-(2-carboxyethyl)cysteine (Fig. 1) upon acid hydrolysis of Hb from acrylamide treated rats. Since, however, we are hypothesizing that glycidamide might be playing a crucial role in the induction of some of the toxic effects associated with acrylamide exposure, there was a need for the development of quantitative methods which would be applicable to both animals and humans and that could be used for the monitoring of both the adduct formed by acrylamide and by its epoxide metabolite. AIMS It was the central aim of this project to develop and apply methods for simultaneously determining the adducts formed by acrylamide and glycidamide in Hb as a means of assessing occupational exposure to acrylamide and to use these data in combination with dosimetric and metabolic studies in experimental animals undergoing toxicological tests to arrive at a risk estimation of human acrylamide exposure which may serve as a basis for regulatory action. ------- \ 0 CH2»CH-C-NH2 Acrylamide Metabolism HB CH-CH2-SH ^«^»^ HB CH-CH2-S-CH2-CH2-C-NH2 HCI I COOH \ / NH, CH-CH2-S-CH2-CH2-COOH /°\ 9 CH -CH-C-NH2 Glycidamide HB CH-CH2-SH OH 0 HB CH-CH2-S-CH2-CH -C-NH 2 HCI I COOH OH \ ' CH-CH2-S-CH2-CH -COOH Z H* O S-(2-carboxycthyl)-cysteinc I der. COOCH3 CH-CH2-S-CH2-CH2-COOCH3 NHCOC3F7 S-(2-carboxy-2-hydroxyethyl)-cysteine I der. I COOCH3 OOCC3F7 CH-CH2-S-CH2-CH-COOCH3 NHCOC3F7 Figure 1. The reactions of AA and GA with cysteine residues in Hb. The products released during acid hydrolysis are 5-(2-carboxyethyl)-cysteine and 5-(2-carboxy-2-hydroxyethyl)- cysteine. The structures of the derivatized products are also shown. ------- In addition to the practical implications of the proposed methods for biomonitoring, acrylamide appears to be a suitable compound for the theoretical development of a risk model based on in vivo dosimetry because of the wide range of toxic effects it is known to induce. Since both acrylamide and glycidamide are reactive electrophilic compounds, it was not clear whether the various toxic effects associated with acrylamide exposure are induced by the parent compound, the epoxide, or the combined action of the two agents. Our finding that acrylamide was metabolized to glycidamide thus generated a few hypothetical solutions to theoretical problems particular to the toxicology of acrylamide. The specific aims of this project were, therefore: 1. To develop a method for measuring the adducts of acrylamide and glycidamide to hemoglobin in rats and to determine both adducts following administration of acrylamide or glycidamide. 2. To develop a method for measuring the adducts of acrylamide and glycidamide in humans, measure adducts in workers occupationally exposed to acrylamide, and correlate adduct levels with neurotoxicity. 3. To evaluate and compare the neurotoxicity and reproductive toxicity of acrylamide and glycidamide in the rat. METHODS ABMl Synthesis of deuterated analogues S-(2-Carbamoylethyl)-3,-3-d2-cysteinewas prepared in analogy with the non-deuterated compound (AACys), the synthesis of which was described in Calleman et al. (1990). Following derivatization with MeOH/HCl and HFBA (Calleman et al., 1978) the mass spectrum obtained upon electron impact ionization gave the fragments m/z = 419 and 205 interpreted as [M]+ and [M - C^CONHD]*, respectively, and the mass spectrum obtained when scanning for negative ions using methane as the reagent gas gave m/z = 418 [M - H]~. Both spectra were in analogy with the nondeuterated compound. TLC, amino acid analysis, and GC/MS revealed an impurity consisting of « 5% cystine. S-(2-Carbamoyl-2-hydroxyethyl)-3,3-d2-cysteinewas prepared as described, in Calleman et al. (1990) for the nondeuterated analogue (GACys). Following derivatization, the mass spectrum obtained upon electron impact ionization gave the fragments m/z = 572 [M - CO2CH3]+, 417 [M - C3F7CONHD]+, and 203 [M - C3F7C02H - C3F7CONHD]+. Chemical ionization using methane as the reagent gas gave the positive ions m/z = 418 [M - C3F7CONHD + H]+ and 632 [M + H]+ and the negative ion 611 [M - HFJ. The ------- spectra were in analogy with the nondeuterated compound. TLC, amino acid analysis, and GC/MS revealed cystine as an impurity of « 5%. On amino acid analysis, three peaks were evident: two peaks, presumably the diastereoisomers (S)- and (R)-S-(2-carbamoyl- 2-hydroxyethyl)-3,3-d2-<#-cysteine (whose presence in approximately equal amounts was indicated by proton NMR spectroscopy), merging into one major peak corresponding to 90%; and a minor peak, possibly the regioisomer S-(l-carbamoyl-2-hydroxyethyl)-3,3-d2- cysteine, corresponding to 10%. The nondeuterated analogue gave the same pattern of peaks but did not contain any cystine. On GC/MS, the isomers were not resolved on the capillary column that was utilized. Treatment of animals Male Sprague-Dawley rats (130-260 g) were obtained from Tyler laboratories and kept on a 12-hr light/dark cycle with free access to food and water. AA (Eastman-Kodak, Rochester, NY) or GA (Polysciences, Inc., Warrington, PA) were dissolved in 0.9% NaCl and administered in a volume of 1 ml/kg body wt by ip injection. Control animals were given vehicle only. Blood was drawn into a heparinized syringe by cardiac puncture under ether anesthesia. Groups of three rats were injected with AA (0, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 mg/kg body wt) and blood was drawn 24 hr after the injection. In an additional experiment, a cumulative dose of 100 mg/kg body wt AA was given in different regimens to groups of four rats. The groups received a single injection of 100 mg/kg, 10 daily injections of 10 mg/kg, or 30 daily injections of 3.3 mg/kg. Blood was drawn 24 hours after the final injection. Isolation of globin and analysis of hemoglobin adducts Erythrocytes and plasma were separated by centrifugation of blood, samples. The red blood cells were washed three times with 0.9% NaCl and lysed by addition of 1 vol of water. Cell membranes and debris were removed by centrifugation at 20,000g for 30 min. The supernatant was added to 10 vol of 1% HC1 in acetone and the resulting precipitate was washed twice with acetone and once with ether and then air-dried. Samples of 30 mg of globin were dissolved in 6 M HC1 to a concentration of 10 mg globin/ml. The deuterated analogues, serving as internal standards for the quantitation, were added to the hydrochloric acid to give concentrations of 10 /xmol/g Hb. The samples were hydrolyzed for 15 hr at 120°C in sealed Pyrex glass ampoules under vacuum (<50 x 10° mm Hg). The hydrolysates were evaporated to dryness, dissolved in 1.5 ml of H2O), and incubated for 1 hr at 37°C. After adjustment of the pH to 9 with NaOH, the samples were applied to Dowex 1 x 8 (100-200 mesh, 1 x 15 cm, HCOO -form) anion-exchange columns. The columns were washed with 80 ml of 5mM formic acid to remove most of the amino acids upon which the adducts were eluted with 60 ml of 100 mM formic acid. ------- The 100 mM eluates were evaporated and derivatized with MeOH/HCl and HFBA. Derivatized samples were dissolved in 0.5-1.0 ml of ethyl acetate before quantitation by GC/MS. Gas chromatography/mass spectrometry analysis The analyses were carried out using a Hewlett-Packard 5890A gas chromatograph linked to a Finnigan 4023 mass spectrometer. The operating conditions for the gas chromatograph were as follows: helium carrier gas at a constant pressure of 15 psi: temperature programmed from 1 min at 70°C, 15°C/min to 230°C, 5°C/min to 240°C, 20°C/min to 280°C, and holding for 1 min. A 30-m DB-5 (0.25 mm i.d., 0.25-^m phase thickness) fused silica capillary column was used. The mass spectrometer was operated in the electron impact mode at an ion source temperature of 250°C and an ionization energy of 70 eV. Chemical ionization mass spectra for the derivatized synthetical adducts were obtained with methane as the reagent gas at an ion source pressure of 0.30 Torr and a temperature of 220°C. Both positive and negative ion spectra were obtained at an ionization energy of 70 eV. For the analysis of the rat Hb samples, 1 /*! of the ethyl acetate solutions was injected directly onto an on-column injector. AACys and its deuterated analogue were monitored at m/z « 202 and 203, [M - QFTCONH^D)]*, respectively, and GACys and its deuterated analogue were monitored at m/z = 202 and 203, [M - QF-jCOJH - C3F7CONH2(HD)]+, respectively. The retention time for AACys was 719 sec and that for GACys was 730 sec. Quantitation was based on the ratio between peak areas of the analyte and the internal standard. Correction for isotopic contributions and nonlinear response (at ratios <0.1) was made with a calibration curve prepared by mixing triplicates of seven rations between zero and one of known amounts of pure nondeuterated standard and the same amount of deuterated standards as added in the rat globin samples. Alkylation of hemoglobin in vitro The rates of reaction of AA or GA with Hb were determined in blood from nonexposed rats. Erythrocytes were separated from plasma by centrifugation, washed with saline, resuspended to the original blood volume using phosphate-buffered saline (PBS), pH 7.4, and incubated with either AA (1 mM) or GA (2 mM) at 37°C. Aliquots were taken from the incubation mixture at different times (5, 10, 15, 20, 40, and 60 min) and the reaction was terminated by adding 5 vol of ice-cold saline followed by centrifugation. The cells were washed twice with saline and lysed by the addition of water. Globin was isolated and the adduct levels were analyzed as described above. ABM 2 Synthesis of alkylated valines N-(-Carbamoylethyl)-L-valine (AAVal) was prepared by dissolving 1.76 g (15 mmole) ------- L-valine in 30 ml H20 and 2.6 ml (18.6 mmole) triethylamine and adding 10 g (141 mmole) AA. The pH of the solution was « 10. After 6 days at room temperature, the product was precipitated with acetone and recrystallized in H2O/ethanol. The product (yield « 32%) was judged to be pure based on TLC, amino acid analysis, 'H-NMR and FAB-MS. The Rrvalue on TLC silica plate, eluted with 2-propanol/H2O 7:3, was 0.65. JH-NMR indicated d 0.95 (6H, dd, val-y), 2.15 (1H, m, val/3), 2.65 (2H, 5, CH2CH2CONHJ, 3.2(2H, m, NHCHjCHj), 3.4 (1H, d, vala). FAB-MS gave the ion m/z = 189 [M+H]+. AAVal was derivatized with methanol/HCl and HFBA according to Calleman et al. (1978) and analyzed by GC/MS. The electron impact ionization spectrum gave fragments m/z = 381 [M]*, m/z = 308 [M-CH2COOCH3]+ and m/z = 212 [M-C3F7]+and chemical ionization with methane as the reagent gas gave the positive ions m/z = 382, 410 and 422 corresponding to [M+l]+, [M+29]+ and [M+41]+. The spectrum was consistent with Structure A in Fig. 2. AAVal was also derivatized with PFPITC according to Mowrer et al. (1986). On GC/MS, the pentafluorophenylthiohydantoin (PFPTH) gave m/z = 395 [M]+ upon electron impact ionization. Chemical ionization yielded the positive ions m/z = 396, 424 and 436 ([M+i;T, [M+29]+ and [M+41]+) and the negative ion m/z = 375 [M-HF]'. See Fig. 2, structure B. N-(2-Carbamoyl-2-hydroxyethyl)-L-valine (GAVal) was prepared in the same way as described for AAVal, but instead of AA, 3.74 g (42.9 mmole) GA was added. The yield was « 44%. The product was judged to be pure by TLC, amino acid analysis, 1H-NMR and FAB-MS. The Rrvalue on TLC was 0.77 in the same system as for AAVal. 'H-NMR indicated 5 0.95(6H, m, val-y), 2.2(1H, val/3), 3.25(2H, m, NHCH2CHOC), 3.5 (1H, dd, vala), 4.4 (1H, m, CHjCHOHCONHz). FAB-MS gave the ion m/z = 205 The product was derivatized with methanol/HCl and HFBA, and run on GC/MS. Five GC-peaks were evident in the approximate ratios 2:1:3:0.7:0.7. Upon electron impact ionization, peak number 2 gave the fragments m/z = 593 [M]*, 242 [M-C3F7]+, 308 [M-CH(OOCC3F7)COOCH3r and 299 Chemical ionization with methane as the reagent gas gave the positive ions m/z = 594, 622, 634 ([M+l]+, fM+29]+ and [M+41]+). The structure of the derivative was consistent with C in Fig. 2. Peaks number 1, 3, 4 and 5 were interpreted as stereoisomers of structure E in Fig. 2. Derivatization with PFPITC gave the PFPTH-derivative, structure D in Fig. 2, which on GC/MS gave the electron impact spectrum with a fragment at m/z = 411 "[M]* while chemical ionization gave the positive ions m/z = 412, 440, 452 ([M+l]+, [M-f 29]+ and [M+41]*) and the negative ion m/z = 391 [M-HF]'. N-(Carbamoylethyl)-dg-L-valine (d8-AAVal) and N-(2-carbamoyl-2-hydroxyethyl)-dg-L- ------- A 9 -I CHj-CH-C-NK, OycMwiMt •C-NHj CHfCH-CM Acrytafltoflt - OH faapUfon tlgleba 1 1 1 . jo*. <*pfpnc \ cc Q. IU cc 0. w a < CO °S^ COOH OH CHEVal ^CH-NHCHj-CHj COOH CEVcl °-O'-CIVCH2-OOOCH, o-c" >^^COOCK, A (CEVlHiFB) C (CHEVaMtfB) O-CH A (CEV«I-KFB) B (AAVaM>FPTM) OH 0 (GAV«M>FPTM) O N- CHjO F (ANV«M>FPTH) Figure 2. The reaction of acrylamide (AA), glycidamide (GA), and acrylonitrile (AN) with the N-terminal valine of hemoglobin in vivo. Following acid hydrolysis of globin, the reaction products of AA and AN with valine are released as CEVal, and the reaction product of GA with valine is released as CHEVal. Compounds A (CEVal-HFB), C(CHEVal-HFB), and E are formed after derivatization with methanol/HCl and HFBA. Following derivatization of globin with PFPITC, the compounds formed are B (AAVal-PFPTH), D (GAVal-PFPTH), and F(ANVal-PFPTH). ------- valine (dg-GAVal) were prepared as the non-deuterated analogues (AAVal and GAVal), but with dg-L-valine as starting material. The syntheses were made on a smaller scale, with a yield of 54% for dg-AAVal and 38% for dg-GAVal. The compounds were pure as judged by TLC. The mass spectra after derivatization with methanol/HCl and HFBA were in analogy with the non-deuterated compounds. NMR spectroscopy and fast atom bombardment mass spectrometry (FAB-MS) Proton NMR spectra were recorded as described in Calleman et al. (1990) and FAB mass spectra were acquired as described in Aim 1. Analysis of Hb adducts in globin hydrolysates Globin was isolated from whole blood as described previously. Samples of 100 mg of globin were dissolved in 6 M HC1 to a concentration of 10 mg/ml. The deuterated reference compounds (dg-AAVal, dg-GAVal, d2-AACys and d2-GACys), serving as internal standards for the quantitation, were added. The concentrations of standards were 1 /xmol/g Hb for the in virro-treated Hb and 100 nmol/g Hb for human samples. The samples were hydrolyzed for 15 h at 120°C under vacuum. The hydrolysates were evaporated to dryness, dissolved in 2 ml H20 and incubated for 1 hr at 37°C. The pH was adjusted to 9 with NaOH and the samples were applied to Dowex 1x8 anion exchange columns (1 x 30 cm, HCOO" -form, 100-200 mesh). The columns were eluted with 150 ml 12 mM formic acid followed by 60 ml 30 mM formic acid, 65 ml 60 mM formic acid and 100 ml 120 mM formic acid. The adduct-containing fractions (see Results) were evaporated and derivatized with methanol/HCl and HFBA. Derivatized samples were dissolved in 100 /xl ethyl acetate from which 2 /*! were injected for quantitation by GC-MS. Analysis of hemoglobin adducts with the modified Edman degradation method Samples of 50 mg of globin were derivatized according to Tornqvist et al. (1988). The amount of PFPITC reagent was increased from 7 to 10 /*!. Globift alkylated with d4-ethylene oxide was used as an internal standard, and was added to give a concentration of 10 nmol/g Hb of N-(d4-2-hydroxyethyl)valine (d2-HOEtVal). The derivatized samples were dissolved in 50 /*! toluene and 1 /*! was injected for quantitative analysis by GC-MS. Gas chromatography-mass spectrometry analysis The analyses were carried out using a Varian 3400 gas chromatograph linked to a Finnigan 4500 mass spectrometer. The operating conditions for the gas chromatograph were as follows: helium carrier gas at constant pressure of 10 psi; temperature programming 100°C - 1 min - 20°/min - 240° - 10°/min - 320° - 2 min for PFPTH-derivatives. A 30 m DB-5 (0.32 mm i.d., 1 fim phase thickness) fused silica capillary column Was used. The mass spectrometer was operated in the electron impact mode at an ion source ------- temperature of 140°C and an ionization energy of 70 eV. In the chemical ionization mode, methane was used as the reagent gas at an ion source pressure of 0.40 Torr. The HFB-derivatives of the valine adducts were analyzed in the positive ion chemical ionization mode. The derivative of CEVal and its deuterated analogue were monitored at m/z = 382 and 388 [M+l]+, respectively, and the derivative of CHEVal and its deuterated analogue were monitored at m/z 622 and 628, [M+29]*, respectively. The retention times for the derivatives of CEVal and CHEVal were 575 and 580 seconds, respectively. Quantitation was based on the ratio between peak areas of the analyte and the deuterated internal standard. The contribution of the deuterated internal standards of the valine adducts to the ions monitored for the non-deuterated compounds was 0.1%. The HFB-derivatives of cysteine adducts were analyzed in the electron impact ionization mode, as described before. The PFPTH-derivatives were analyzed in the negative ion chemical ionization mode. AAVal-PFPTH was monitored at m/z = 375 [M-HFJ and d^HOEtVal-PFPTH at m/z = 352 [M-HF]\ In addition to the valine adduct of AA, N-(2-cyanoethyl)valine-PFPTH (ANVal-PFPTH, Fig. 2 structure F), the valine adduct of AN, was monitored at m/z = 274 [M-103]'. The retention times for the PFPTH-derivatives of AAVal, ANVal and d4-HOEtVal were 826, 701 and 692 seconds, respectively. The quantitation was based on the ratio between peak areas of the analytes and d4-HOEtVal. Alkylation of hemoglobin in vitro The rates of reaction of AA or GA with N-terminal valine in human Hb were determined in blood from a non-exposed person, as described earlier. The incubation was made with AA (380 mM) or GA (100 mM) and aliquots were taken at different times (0-40 min.). The adduct levels were determined in hydrolyzed globin samples as described above. Calibration The analyses of PFPTH-derivatives were calibrated as follows: A calibration curve was prepared by addition of different amounts of in vitro alkylated (with AA or AN) globins, to 50 mg globin samples from a non-exposed person. The alkylation levels were 5-80 nmol/g Hb. d4-Ethylene oxide alkylated globin was added to give 10 nmol d4-HOEtVal/g Hb. The calibration curve for AA was slightly concave and for AN linear in the range studied. Calibration samples were run every day when unknown samples were analyzed in order to compensate for variations in sensitivity of the mass spectrometer. The adduct levels of the in vitro alkylated globins were determined after acid hydrolysis and ion-exchange chromatography by the procedure described above. For the AA-globin, dg-AAVal was added as internal standard. For the AN-globin, which was alkylated with I4C-AN (168 Bq/nmol), the radioactivity in the peak corresponding to CEVal was counted following ion-exchange chromatography. 10 ------- Field study in the People's Republic of China Physical description of the factory f The acrylamide shop selected for this study was part of a large chemical factory employing 700 individuals in the city of Xinxiang, Hunan Province, China. The production of acrylamide, which had started in 1982, was operated in three shifts and amounted to 1,000 tons/year. The acrylamide production line operated as follows: acrylonitrile (bought from an external supplier) was converted to acrylamide (20% solution) by catalytic hydration with metal catalysts. The latter solution was then concentrated to give a 35% acrylamide solution, which was purified by cation and anion exchange. This solution is stored in large vessels and then transferred to smaller barrels: all these processes are performed in the same room, the "synthesis room." Barrels are then moved to an adjacent room, the "polymerization room," where acrylamide is mixed with acrylic acid, starch (35% of final product) and sodium allylsulfonate to generate a polyacrylamide co-polymer. After grinding and drying, the co-polymer is then bagged in another adjacent building, "the packaging unit." The use of the copolymer is primarily in the oil industry to fill the drilling tube and decrease friction and to harden soil and allow extraction of compact samples. Air monitoring The air concentrations of acrylamide were monitored twice during the summer months and twice in the month of September of 1991 when the study was undertaken. Each time several determinations were made. Acrylamide was sampled for 10 minutes (flow rate 3 1/min) by station sampling and determined by gas chromatography with a FFAP column and an electron capture detector following bromination. The detection limit of this method is 0.5 ng of acrylamide, equivalent to an air concentration of 0.03 mg/m3 under the testing and sampling conditions used. The results of these analyses are shown in Table la. In the summer the air concentrations ranged from 0.3-8.8 mg/m3 with an extreme value of 153.1 during the discharge of acrylamide (not included in Table la), whereas during the month of September when specimens for the determination of biomarkers were collected, a relatively narrow range of air concentrations of 0.11-1.64 was determined. Air levels of acrylonitrile were also monitored in June 1991 using a charcoal sampler and gas chromatography analysis (Table Ib). Description of study populations A total of 41 workers, 34 males and 7 females, occupationally exposed to acrylamide while working in the processes of synthetization, condensation, filling, polymerization, and maintenance, etc. were selected for the study. Depending on the primary location where 11 ------- Table 1 la. Air concentrations of acrylamide (mean ± SD, mg/m3). Summer Autumn Average Polymerization room 5.95 ± 2.55 (n = 6) 0.58 ± 0.21 (n = 6) 3.27 ± 3.29 (n = 12) Synthesis 1.65±0.89(n = 8) 0.61 ± 0.55 (n= 10) 1.07±0.88(n=18) Ib. Air concentrations of acrylonitrile (mean + SD, mg/m3) Summer Polymerization room 8.4±3.7(n = 6) Synthesis room 44.7±37.9(ir=6) 12 ------- these workers performed thek work, they were classified into synthesis workers, polymerization workers, packaging workers and ambulatory workers which included foremen, repairmen, etc. Two individuals who had only minimal exposure to acrylamide during the preceding 4- month period and two others who had been employed in the workshop for less than half a year, and thus were not expected to have had time to develop neurotoxic symptoms, were excluded from the regression analysis. The ages of the workers ranged between 18 and 42 years (mean 27 years). The exposure duration was 3 years on average with the shortest being one month and the longest 9 years. Ten workers had been exposed to acrylamide for more than 5 years. Thek personal protection measures included gauze masks, cotton clothes, rubber gloves and boots. The workers were brought into the hospital ward approximately 1 hr after they had started the morning shift. Blood was collected immediately upon arrival and the workers remained in the ward for medical examination and collection of urine for a 24-hr period. For the Vibratron measurements the reference group consisted of 105 unexposed and healthy adults (51 males and 54 females ages 20-60 years) including mechanical workers and office personnel and for the ENMG a historical control group of 80 persons was used. Persons with diabetes and nervous diseases were excluded from both groups. For the biomarkers of exposure and signs and symptoms of neurotoxicity, ten unexposed individuals from the same city as the exposed group were studied. Determination of mercapturic acids in urine Urine samples were collected during 24 hrs and the volumes and specific gravities were determined. 1 ml of these samples was hydrolyzed with 1 ml of 6N HC1 at 85°C for 4 hr. Under these conditions the N-acetyl group of the mercapturic acid as well as the amide group was hydrolyzed to give S-(2-carboxyethyl)cysteine. After centrifugation at 3,000g for 10 min, 0.1 ml of supernatant hydrolysate solution was evaporated to dryness under N2 at 65°C. The residue was dissolved in 1 ml of water. 200-/*1 aliquots of the hydrolysate were mixed with 100 j*l of the fluoraldehyde reagent. After 1 min, 10 fd of sample was subjected to HPLC. Urine samples were acidified with 1% HC1 and frozen, and kept at -20°. Solvent A was tetrahydrofuran-methanol-0.1 N sodium acetate (5:95:900, pH 7.2). Solvent B was methanol-water (9:1). The column was kept at ambient temperature, and 10 (A of standard or sample were injected into the column. Elution was achieved by running a gradient of 10% solution B over 8 min at a flow rate of 1.5 ml/min, then flashing the column with 90% solvent B at the end of each injection to eliminate the less polar compounds in urine. Blood samples for hemoglobin adducts The blood was drawn in vacutainer tubes containing EDTA as anticoagulant. Workers 13 ------- were involved in the production of AA (synthesized by catalytic hydration of AN) and polyacrylamide, and had been exposed to AA and AN for 0.1 to 8 years. Blood samples were also collected from 10 control workers in the same city, who had not been exposed to AA or AN. Measurements of vibration thresholds A Vibratron n vibration sensitivity tester was used for the quantitative measurements of vibration threshold of bilateral index fingers and big toes of each subject in both groups at a room temperature of 22-25°C. The instrument consisted of two modules with vibrating posts and a controller. The testing procedure was a two-alternative forced choice procedure. While the position of vibration and intensity sequence were under control, the subjects were asked to press their fingers or toes against each rod and to determine which of the two rods was actually vibrating. The vibration settings of the five errors and the five lowest correct scores were then determined. The highest and lowest values of the ten scores were eliminated and the mean of the remaining eight scores determined the vibration threshold (1 vibration unit (VU) = 0.5 microns peak-to-peak amplitude). Electroneuromyography (ENMG) The ENMG was carried out with a Dantec 2000C electromyographic apparatus. The electric activities of the abductor pollicis brevis, abductor digit! minimi and anterior tibialis muscles, including insertion activity, spontaneous activity of motor units, and electrical activity during mild and maximal voluntary contraction, were recorded with concentric needle electrodes. The mean duration and mean voltage of 20 motor unit potentials and the percentage of polyphasic potentials in each muscle were determined. The duration of a motor unit was taken as the time for the oscilloscope tracing to return to the base line after its original deflection from the beginning of that potential. The voltage of a motor unit was taken as the peak-to-peak amplitude. The phase of a motor unit potential was defined by the number of deflections across the base line. The minimum acceptable skin temperature of the hands and feet during the measurements of nerve conduction velocity and distal latency was 30°C. Bipolar skin stimulation electrodes were used in all the measurements of nerve conduction velocity. The voltage was always 30% supramaximal. For measuring maximal motor nerve conduction velocity (MCV), the median and ulnar nerves were stimulated at the elbow and the wrist, and the muscle action potential was recorded from the abductor pollicis brevis muscle and the abductor digiti minimi muscle with concentric needle electrodes. The peroneal nerve was stimulated at the knee and ankle, and the muscle action potential was recorded from the extensor digitorum brevis muscle by surface electrodes. The sensory nerve conduction velocity (SCV) of the median and ulnar nerve was measured through the stimulation of the forefinger and the little finger, respectively, with ring electrodes. The sensory nerve conduction velocity of the sural nerve was recorded with a 14 ------- surface electrode at midcalf, 14 cm from the stimulating electrode placed just below the lateral malleolus. The peak-to-peak amplitude of the evoked potentials was also measured. The normal values of all related ENMG parameters were previously established in our laboratory using the same EMG apparatus and the same technique in 80 unexposed healthy adults. Clinical and laboratory examinations Physical and neurological examinations were conducted in both groups according to a protocol previously described (He et al. 1989). The laboratory studies included routine blood and urine tests, liver function (serum glutamate pyruvate transaminase and the thymol turbidity test) and serum hepatitis B surface antigen. Questionnaire information All subjects were interviewed with the aid of a structured questionnaire collecting information on demographic factors, smoking and drinking habits, height and weight, occupational history, past illnesses, reproductive history and family history. Definition of Neurotoxicity Index A neurotoxicity index (NIn) with a maximal score of 50 points was defined so as to provide a quantitative parameter expressing the peripheral neuropathy which could be correlated to the different biomarkers of exposure. The different observations scored in the Neurotoxicity Index are shown in Table 2. Clarifications for this Table include the following: (1) workers who had lost their pain or touch sensation got 1 to 3 points depending on the extent of this loss in fingers, hands, or forearms, (2) up to 2 points each for increased vibration thresholds in hands or feet as measured with the Vibratron instrument were given. Vibratron thresholds were compared to the corresponding control group with regard to age. If their ratios to the corresponding controls were 0-4 for toes or 0-2.5 for index fingers, a score of 1 point was given for each, whereas if the ratios were 4-8 or 2.5-5 for toes or fingers, respectively, 2 points were given for each, (3) for each observation of an ENMG abnormality 0.5 points were given, with a maximal possible total of 12 x 0.5 = 6 points. Statistical methods The chi-square test was used to analyze the symptoms and signs, and the Student's t-test was used in the analysis of ENMG parameters. The variance analysis and Q-test (Neuman-Keuls methods) were used in the comparison of vibration thresholds between the reference group and the acrylamide group, both with different age categories. Univariate and multivariate regression analysis was used to estimate correlation coefficients and levels of statistical significance for the relationship between the Neurotoxicity Index and the 15 ------- TABLE 2 Definition of Neurotoxicity Index Points Numbness of extremities 1 Cramping pain 1 Loss of position sensation 2 Loss of pain sensation 0-3 Loss of touch sensation 0-3 Loss of vibration sensation: According to tuning fork 1 Vibration threshold in big toe 0-2 Vibration threshold in index finger 0-2 Clumsiness of hand 4 Difficulty grasping 4 Unsteady gait 4 Muscular atrophy 6 Decrease or loss of ankle reflexes 3 or 5 EMG 6 ENMG 0.5 per abnormality (max 6) Maximum £ 50 16 ------- biomarkers and ANOVA analysis was used for comparing the means of the different categories of workers. AIM 3 Animals and treatments Male Sprague-Dawley rats weighing 270-3 lOg were divided into five groups and allowed free access to food and water. Rats were injected ip with AA or GA once a day for 8 days. The doses were 25 and 50 mg/kg for AA and 50 and 100 mg/kg for GA. Control rats were injected with the vehicle, distilled water (1 ml/kg). All animals were tested and sacrificed on day 9, 24 hr after the last injection. For the reproductive toxicity experiments, sexually mature Sprague-Dawley rats (350g) were utilized. Animals were administered either AA (50 mg/kg/day for 7 days) or GA (50 mg/kg/day for 14 days). Neurotoxicity testing Rotarod. The rotarod apparatus consisted of a horizontal plastic rod (6 cm in diameter) positioned 30 cm above a switch floor. The rod was divided by partitions so that 4 rats could be tested simultaneously. All rats were trained to stay on the rod (rotating at 20 rpm) twice daily for 5 consecutive days prior to the beginning of treatment. The cut off-time was 100 sec. The test was repeated 3 times at 20-min intervals and the average value of the measurements (in seconds) was utilized. Hindlimb Splay. This method was similar to that described by Edwards and Parker (1977). Each rat was held in a horizontal position with the dorsal side up, 32 cm above a flat surface which was covered with a thin layer of sawdust. The rat was then dropped and the position of the fourth digit of each hindlimb on landing was marked and the distance between the toe(s) marks was measured. Values represent the average of 3 consecutive measurements. Reproductive toxicity testing Twenty-four hours after the last injection, animals were sacrificed by cervical dislocation under ether anesthesia and testis, epididymis and vas deferens were dissected and weighed. The head, body and tail of epididymis were minced and homogenized and the number of spermatozoa was determined. Sperm viability was measured as described by Bishop and Smiles (1957). 17 ------- RESULTS AND DISCUSSION AIM1 Development of a GC/MS method for measuring Hb adducts of AA and GA The reactions of AA and GA with cysteine residues of hemoglobin are shown in Fig. 1. Globin isolated from rat blood was subjected to acid hydrolysis and the adducts were released as S-(2-carboxyethyl)-cysteine and S-(2-carboxy-2-hydroxyethyl)-cysteine. The use of anion-exchange chromatography for purifying these particular adducts from the neutral and basic amino acids takes advantage of their extra carboxylic acid function. The hydrolysate was applied to the column and the most negatively charged amino acids were most strongly attached. With weak formic acid (5 mM) all amino acids with only one carboxylic acid group were eluted with the front at 5-20 ml, while 100 mM formic acid eluted the more strongly bound dicarboxylic acids. Since, however, both glutamic acid and aspartic acid are dicarboxylic acids, the 100 mM eluate contained, in addition to the two adducts, part of the glutamic acid content and all of the aspartic acid. The elution volumes were 70-90 ml for glutamic acid, 90 ml for AACys, 95-110 ml for aspartic acid, and 90-110 ml for GACys. After evaporation, the 100 mM eluates were derivatized for quantitation with GC/MS. Multiple ion monitoring in the electron impact ionization mode was chosen because with the instrument used it provided a higher sensitivity than the negative ion chemical ionization mode. The fragments monitored for the internal standard had one deuterium instead of the two deuteriums in the parent molecule due to loss of 1 deuterium atom in the fragmentation process (Calleman et al. 1990). The difference in m/z of only 1 between the analyte and the internal standard is not ideal since the contribution from the deuterated ion to the nondeuterated was 1% and the contribution from the nondeuterated ion to the deuterated was 10%. However, this was corrected for by use of the calibration curve. The use of free deuterated amino acids as internal standards was based on the assumption that there is no preferential decomposition during acid hydrolysis of an amino acid depending on whether it is free in solution or exists as a peptide residue. Studies by Crestfield et al. (1963) on the close structural analogue S-carboxymethyl-cysteine in ribonuclease showed that 97, 103, 103, and 101% of this amino acid were recovered after 24, 48, 73, and 102 hrs of hydrolysis in 6 M HC1, respectively. The fact that the concentration of S-carboxymethyl-cysteine did not decrease after 24 hr when it existed free in solution indicates that this amino acid is equally stable as a free amino acid and as a peptide residue. The synthetic standards of AACys and GACys for preparation of the calibration curve were quantified by amino acid analysis employing the response factor of leucine. The compounds contained no impurities. The detection limit for the GC/MS was 1.7 pmol of injected derivatized standard compound. The calibration curve on GC/MS was linear between the ratios of 0.1 and 1.0 with R2 = 0.999 for both derivatized AACys and GACys. 18 ------- Analyses in triplicate gave an average coefficient of variation (CV) of 6% for AACys and 8% for GACys. Despite the fact that deuterated internal standards were used, the GC/MS system exhibited a certain drift in the ratios determined between different occasions on which it was used. Thus, while one globin sample was processed and analyzed several times, the CVs for AACys (n=4) and GACys (n=5) were 2 and 4%, respectively, on a single occasion; the CV for repeated (n=5) injections of an identical sample on a different occasion was 2% for AACys and 13% for GACys. The CVs within the dose groups of rats were on average 13 and 15% for AACys and GACys, respectively. Background levels found in the globin from control rats were 0.15 (0.14-0.17) /xmol/g Hb and 0.015 (0.010-0.019) /xmol/g Hb for the AA and GA adducts, respectively. Administration of 1 mg/kg body wt acrylamide by ip injection to rats resulted in a statistically significant increase in adduct levels (p<0.01). Acrylamide and glycidamide hemoglobin adducts following administration to rats In a first experiment, rats were injected with different doses of AA and the adduct levels (AACys) in the blood were measured 24 hr after injection (Fig. 3). At this time all AA presumably had reacted since no parent compound was detectable by Miller et al. (1982) in any tissue after 1 day. The AA adduct level increased approximately linearly with the administered amount. The levels of the GA adduct (GACys), on the other hand, produced a concave curve (Fig. 3b). A typical mass fragmentogram from a rat treated with 10 mg/kg body wt of AA is shown in Fig. 4. In a second experiment rats were administered increasing doses of GA. In this case the GACys adducts increased linearly with the administered amount (Fig. 5). On a mol/kg body wt basis, levels of GACys adducts were 3.2 times lower than the AACys levels measured following AA administration. From these experiments, the percentage of the initial concentration of AA metabolized to GA was calculated (Fig 6). For this purpose, the concentration of GA (in mmol/kg) corresponding to the GACys adduct level in the AA-injected rats was calculated as if GA had been injected directly. This concentration was divided by the initial concentration of injected AA. At the lowest concentration of AA (5 mg/kg body wt) about 51% was converted to GA, while at the highest concentration (100 mg/kg body wt) only about 13% was metabolized to GA. In an additional experiment, a cumulative dose of 100 mg/kg body wt AA was given as a single dose, distributed over 10 consecutive days as 10 mg/kg/day, or distributed over 30 days as 3.3 mg/kg/day. Twenty-four hours after the final injection, blood was drawn and the adduct levels of both AACys and GACys were measured. Table 3 shows the measured adduct levels and, for comparison, the expected levels calculated according to Eq. (1): 19 ------- z o> o 3. 0» t> 0 •o < 10- 8- 6- 4- 2- 0 J 20 40 60 80 Acrylamide (mg/kg bw) 20 40 60 80 Acrylamide (mg/kg bw) 100 Figure 3. Hb cysteine adduct levels of AA and GA in rats injected with different single doses of AA.(n) Cysteine adduct of AA; (*) cysteine adduct of GA. Symbols represent means +SD of three to five animals, (a) Full scale, (b) Enhanced detail of (a). 20 ------- 202 . 283 . 285 . 785 GACys 0.12 nmol/g Hb 738 •A internal standard for GACys =F= 716 7J9 AACys 0.79 nmol/g Hb 729 718 internal standard for AACys 729 725 738 735 746 19520. 977928. 376328. 2453588. 745 S Figure 4 . A typical mass fragmentogram from a rat treated with 10 mg/kg body wt AA. The y-axis shows the intensities of the ions monitored, m/z = 202, 203, 204, 205, for the derivatized nondeuterated and deuterated forms of S-(2-carobxy-2-hydroxyethyl)-cysteine and S-(2-carboxyethyl)-cysteine. The x-axis shows retention time in seconds. 21 ------- o 3L «> O •o •o 2.5- « 2.0- 1.5- 1.0- 0.5 0.0 20 40 60 80 100 Glycidamide (mg/kg bw) Figure 5. Hb cysteine adduet levels of GA in rats injected with different single doses of GA. Symbols represent means +, SD of three animals. 22 ------- o o o> > c o 100- 90- 80- 70- 60- 50- 40- 30- 20- 10- 0 20 40 60 80 100 Acrylamide (mg/kg bw) Figure 6. Conversion of AA to GA in vivo in rats injected with AA. The percentage is calculated in relation to the initial concentration of AA in the body. The fraction converted to GA is estimated from measured adduct levels of GA. 23 ------- TABLE 3 Hemoglobin adducts in rats treated with different dose regimens of acrylamide Measured adduct level Dose rate Expected adduct level0 (mg/kg body wt) (|imol/gHb) (SD, n) (jimol/gHb) 1 Day x 100 10 Days x 10 30 days x 3.3 AACys* 8.62 GACysc 0.364 AACys 5.72 GACys 1.00 AACys 3.58 GACys 0.749 (0.66,5) (0.049, 5) (0.99,4) (0.17,4) (0.45,4) (0.062, 4) AACys 8.62 GACys 0.364 AACys 5.60 GACys 0.96 AACys 4.20 GACys 0.79 a The expected values were estimated from the data shown in Fig. 3 and were corrected for the replacement of old erythrocytes and the increase in blood volume due to the growth of the animal (Eq. (1)). The values of a in Eq (1) were 3.3 mg/kg/day, 0.23 and 0.044 iimol/g Hb for AA and GA, respectively, and, at 10 mg/kg/day, 0.645 and 0.1095 jimol/g Hb for AA and GA, respectively. The body weights increased from 132 to 380 g during the 30-day treatment and from 264 to 296 g during the 10-day treatment. The measured adduct levels were not significantly different (p > 0.05) from the expected adduct levels. b AACys = The Hb cysteine adduct of acrylamide. c GACys = The Hb cysteine adduct of glycidamide. 24 ------- = E o(l - t) x ft, (1) t=l I. where {[RYMY]},^ is the accumulated level of adducts at the end of exposure, a is the daily increment of adducts based on the measured levels in the first experiment (dose-response of AA), t,, is the duration of the experiment in days, and t^is the lifetime of the erythrocytes estimated to 61 days in the Sprague-Dawley rat (Derelanko, 1987). The factor b in the equation (body wt at time t^+i/body wt at tj is used for correcting for the increase in total blood volume due to the growth of the animal which will cause a dilution of the adducts. This equation for the accumulation of Hb adducts during chronic exposure is based on the assumption that the Hb adducts are eliminated at the same rate as the erythrocytes. The measured adduct levels were not significantly different than the expected adduct levels. Levels of AA adducts decreased as the dose rate decreased mainly because of the elimination of old erythrocytes and dilution of adducts due to body growth, but also because of a larger fraction of AA metabolized to GA. In fact, levels of GA adducts increased when the same cumulative dose of AA (100 mg/kg) was given as ten 10-mg/kg doses over a 10-day period, suggesting higher conversion of AA to GA. When the same amount of AA (100 mg/kg) was given over 30 days, however, although levels of GA adducts were higher than after the acute 100 mg/kg dosage, they were not as high as at the 10 x 10- mg/kg dosage regimen because of elimination of old erythrocytes and body growth. The constants k^, and kt, as well as t1/2 are presented in Table 4. The second-order rate constants, k^,, for the reaction between cysteine residues in hemoglobin and AA or GA were determined in vitro. These rate constants were calculated from k^. = [RY]/[Y]/D, where [RY]/[Y] is the degree of alkylation measured in the experiment, D is the dose which may be calculated from D = [RX] x t, and [RX] is the concentration of AA or GA in the incubation mixture (Segerback, 1990). The first-order rate constants for the elimination of AA and GA in the blood compartment, kj,, were estimated from adduct levels of AA or GA, when AA or GA was injected directly, using Eq. (2): ' where [RX]0 is the initial concentration of the injected compound. Since AA distributes rapidly and evenly in the body to all tissues (Miller et al. 1982), its initial concentration may be estimated on the basis of 1 kg body wt = 1 liter; the same assumption is also made for GA since it is a highly water-soluble low-molecular-weight molecule. The k^, 25 ------- TABLE 4 Pharmacokinetic parameters of acrylamide and glycidamide in the rat Compound Acrylamide Glycidamide a Second-order kcy$fl (liter/g Hb x hr) 1.8x10-3 0.92 x 10-3 rate constant for the reaction ke.* (hr') 0.37 0.48 with cysteine in rat Hb in tl/2c (hr) 1.9 1.5 vitro. b Estimated first-order rate of elimination in the blood (Eq. (2)). c Half-life in vivo in the blood. Calculated from i\n = In2 26 ------- determined in vitro are used in Eq. (2) since the rates of reaction of AA or GA with Hb-cysteine in vivo are assumed to be the same as in vitro. The ratio [RY]/[Y] was taken from in vivo data assuming complete reaction of the parent compound within 24 hrs. The half-life is calculated from the relationship t1/2 = In 2/lg. General discussion of Aim 1 The spectrum of different amino acids in Hb which react with AA and GA is currently not known. There are several different nucleophilic amino acids such as histidine, cysteine, and N-terminal valine in hemoglobin (EPA, 1990) which may give rise to adducts useful for monitoring exposure to these electrophiles. The unsaturated bond of AA is, however, known to have high reactivity toward -SH groups in Michael type additions, which made cysteine adducts a natural choice for the development of our quantitative technique along the lines of the work of Bailey et al. (1986). Although the epoxide GA is expected to react significantly also with nitrogen- and oxygen-containing nucleophilic amino acids, the simultaneous determination of adducts formed by both agents provided an analytical procedure with the appeal of simplicity. Thus, a method was developed to determine the cysteine adducts formed by the two electrophilic agents. The adduct levels of AA in our study are in good agreement with those determined by Bailey et al. (1986) in the concentration range 0-5mg/kg. The background level of AACys in that study, however, was 7 times lower than the background levels determined in our study (0.15 /xmol/g Hb), which might be explained either by the use of different strains of rats (Wistar vs Sprague-Dawley) or by methodological differences, such as the use of different internal standards for quantitation. The pronounced concavity of the GA adduct curve in rats injected with AA indicates that this metabolite is formed by a saturable process following Michaelis-Menten kinetics. It should be noted that because of our interest in providing 'data useful for relating hemoglobin adduct levels to neurotoxic effects, the dose range of injected AA and GA is rather high, compared to most previous studies of Hb adducts with other compounds. Thus, nonlinearities in the curve shapes, rarely observed at low doses, become evident. As discussed previously (Calleman et al., 1990), the most likely candidate for the enzyme system responsible for the metabolic conversion of a simple alkene such as AA to GA is cytochrome P-450, although this hypothesis has not yet been tested. The saturable kinetics of this process may provide a possible explanation for the fact that AA has consistently given a negative response in the Ames/Salmonella test also in the presence of S-9 mix. Indeed, the case of AA could be similar to that of ethylene and propylene which are negative in the Ames test while their active metabolites, ethylene oxide and propylene oxide, respectively, are clearly mutagenic. It has been proposed that the negative responses of the two parent alkenes are due to the fact that their saturable metabolism may limit the maximal dose of epoxide generated in the test system to a level below what is required for a statistically significant response (Osterman-Golkar and Ehrenberg, 1982). 27 ------- The experiments performed show that direct administration of GA results in the formation in vivo of the same adduct, S-(2-carboxy-2-hydroxyethyl)-cysteine, as that previously reported as evidence for the formation of this epoxide in AA-treated animals (Calleman et al. 1990). Adduct formation in GA-treated animals appears to be a linear function of the injected dose. The origin of the background levels of adducts is not known. It is unclear whether they are produced as methodological artifacts or if they reflect true adduct levels. Since the adducts measured do not contain the amide group originally present, the adducts are not entirely specific to AA (or GA) exposure. For example, exposure to acrylonitrile has been shown to give S-(2-carboxyethyl)-cysteine in hydrolyzed Hb (Fennell et al., 1989). Intermediates in glycolysis, such as 3-phosphoenolpyruvate, 3-phosphoglycerate, and 1,3-diphospho- glycerate could theoretically give rise to S-(2-carboxy-2-hydroxyethyl)-cysteine following protein hydrolysis. S-(2-carboxy-2-hydroxyethyl)-cysteine (Ohmori et al. 1965) and S-(2-hydroxy-2-carboxyethyl)-cysteine (Yao et al. 1970) have been found in the urine of humans not known to be exposed to electrophilic substances. This indicates that the formation of the background levels of these specific adducts may be a result of endogenous processes. Background levels in humans and rodents of several other low-molecular-weight adducts have been found. The hemoglobin binding index for AA to cysteine (6400 pmol (g HbWmol (kg body wt/1, is higher than for any other compound studied in the rat and is higher than that of GA (1820 pmol (g Hb/V^mol (kg body wt)'1), calculated at 50 mg/kg body wt of AA and GA, respectively. This difference is primarily explained by a lower reactivity of GA toward Hb-cysteine and a shorter half-life (t,/^ in blood, compared to that of AA, thus giving a lower yield of adducts. The half-life for AA (1.9 hr) and GA (1.5 hr) reflects the sum of all reactions leading to the elimination of the compounds from the blood and is estimated on the assumption of a linear rate of elimination (Eq. (2)) in the concentration range studied. The half-life of AA is given as an average over the dose range 0.5-100 mg/kg body wt, although the elimination rate is expected to decrease with increasing administered doses of AA due to the saturable formation of GA. The half-life of AA in blood determined by Miller et al. (1982) and by Edwards (1975) was 1.7 and 1.9 hr, in good agreement with our data. The second-order rate constant for the reaction of AA with Hb was determined by Hashimoto and Aldridge (1970) to be 17 x Ifr31 x (g Hb x hr)'1 [as calculated from a reported value of 18 1 x (mol x min)'1], as compared to 1.8 x 10'31 x (g Hb x hr)'1 in our experiments. The experimental conditions, however, were not identical in the two studies. In particular, the pH used by these authors was 8.0, higher than that used in our experiments (7.4); this is expected to increase the rate of reaction with cysteine residues because of the increased thiolate ion concentration at higher pH. Possibly the most notable result of the present study is the high extent of metabolic conversion in vivo of AA to GA, reaching more than 50% at the lowest dose studied. As shown in Fig. 6 there is a higher percentage of AA converted to GA at lower doses of AA. Consequently, if a toxic effect induced by AA is due to GA, the relative importance of the 28 ------- risk for this toxic effect will increase at a lower AA exposure. For risk estimation, it will be, therefore, necessary to investigate which of the toxic effects induced by AA might be related to the parent compound, to the metabolite, or to a combination of the two. It is noteworthy that an injection of 50 or 100 mg/kg body wt AA will give the same tissue dose of GA as an injection of 10 mg/kg body wt GA. Thus, for a toxic effect which is exclusively caused by GA, we would expect the same effect if we inject 50-100 mg/kg body wt AA or 10 mg/kg body wt GA. This is based on the assumption that the tissue dose is directly related to the toxic effect in a dose-response relationship. When AA was given in fractionated doses (i.e., in the chronic exposure regimen), a higher proportion of the cumulative tissue dose consisted of GA than after high-dose acute exposure (Table 3). Consistent with the results from the acute exposure experiments, this suggests that the risk for toxic effects caused by GA might be of greater importance at lower dose rates of AA in long-term exposure situations. In this experiment, the measured adduct levels were in good agreement with those calculated using the adduct levels from single injections of AA, suggesting that these particular adducts may be eliminated at the same rate as the erythrocytes. They appear, therefore, to be stable and useful for determination of tissue doses. In our calculation of expected values of Hb adduct levels, we have corrected not only for the elimination of old erythrocytes, but also for the dilution resulting from the increased body weight (Eq. (1)), and hence blood volume, during the course of the experiment. The apparently nonlinear decrease of Hb adduct levels often observed in rats following acute, single treatments of electrophilic agents (Neumann, 1981) may thus be explained by an increased blood volume during the course of the experiment rather than by a lack of stability of the adducts. AIM2 Development of methods for acrylamide and glycidamide Hb adducts in humans Methods were developed for the determination of AA and GA adducts to N-terminal valine, both following total hydrolysis of the protein and by means of the modified Edman degradation procedure (Fig. 2). In the first method, globin isolated from human blood was subjected to acid hydrolysis. In this procedure the amide groups originally present in AA and GA were converted to carboxylic acid groups during acid hydrolysis and thus valine adducts were released as N-(2-carboxyethyl) valine (CEVal) and N-(2-carboxy-2-hydroxyethyl) valine (CHEVal). The hydrolysates were applied to anion exchange columns and eluted stepwise with increasing concentrations of formic acid according to a modification of the method utilized in rats. Most of the normal amino acids, including glutamic acid, were eluted with 150 ml 12 mM formic acid. CECys eluted in the 60 ml 30 mM fraction, aspartic acid eluted in the 65 ml 60 nM fraction. CEVal eluted partly in the 30 mM fraction and partly in the 60 nM fraction, and these fractions were therefore pooled. The 100 ml 120 mM fraction contained 29 ------- CHECys and CHEVal. The adduct-containing fractions (30 mM + 60 mM and 120 mM) were evaporated and derivatized with methanol/HCl and HFBA for GC-MS. The second-order rate constants for the reactions of AA or GA with N-terminal valine, kvj, and key,, in human Hb determined in vitro are presented in Table 5. In human Hb, AA is about 3 times more reactive towards cysteine than versus N-terminal valine, whereas GA is about equally reactive towards these two sites. The rate constant for the reaction of GA with N-terminal valine in Hb is comparable for human and rat. In contrast, the reactivity of cysteine in human Hb is 2 orders of magnitude lower than in rat Hb. This is explained by the presence of a reactive cysteine in position 125 of the b-chain in rat Hb, which is lacking in this position in human Hb (Hamboek et al., 1981). Symptoms and signs in acrylamide exposed workers The percentage of workers displaying different kinds of symptoms and signs are shown in Table 6. Fatigue (71%), numbness in hands and feet (71%), sweating (68%) of hands and feet and peeling of skin (59%) were the early and prominent symptoms in the acrylamide group and were much more frequently seen than in the reference group. The peeling of the skin in hands usually appeared within 2-30 days after topical contamination by aqueous acrylamide. Unsteadiness of grasping and walking were present in 6 and 10 acrylamide workers, respectively. Systemic symptoms such as dizziness, nausea, insomnia and headache were also found but their prevalences were not significantly different from controls. On neurological examination, impairments of pain (54%), touch (46%) and vibration (41%) sensation were prominent in the distal part of lower limbs and hands of the exposed workers. Diminution or loss of ankle reflexes were found in 29% of the acrylamide group. There were 6 and 8 acrylamide workers having difficulties in standing on one leg and displaying a positive Rhomberg sign, respectively. None of these abnormalities were seen in the reference group. No cerebellar involvement was found in either group. The results of a survey on the reproductive history of each subject showed no difference in fertility, abortion and birth defects in offspring between the acrylamide workers and referents. Vibration thresholds The vibration thresholds of index fingers and big toes in the reference group showed no difference between the two sides, nor between the two sexes (data not shown). However, the difference in vibration thresholds was significant among the 3 age-subgroups of referents, namely, < 31 years, 31-40 years, and > 41 years (p<0.05), showing an elevation of vibration thresholds along with the increase of age in unexposed healthy adults. A significant increase of vibration thresholds was found in acrylamide workers of all age groups (Table 7). 30 ------- TABLE 5 Second-order rate constants for the reaction of acrylamide and glycidamide with amino acid residues in hemoglobin [liter (g Hb)'1 hr1] x 106 Compound Acrylamide Glycidamide Human *Val *Cys 4.4 14 11 9.5 Rat *Val N.D." 12* kCys< 1800 920 a Not determined b GAVal was determined in globin from a rat treated with 100 mg/kg GA and Jh/al was calculated from: Aval = (GAVal x JtCys)/GACys. c Data are taken from Table 4. 31 ------- TABLE 6 Percentage of acrylamide-exposed workers and controls with symptoms of adverse health effects Numbness of extremities Fatigue Sweating of hands and feet Skin peeling Menstruation disorders Loss of pain sensation Dizziness Anorexia Loss of vibration sensation Nausea Loss of ankle reflexes Insomnia Headache Unsteady Gait Loss of knee jerk Unsteady Rhomberg sign Loss of triceps reflexes Loss of biceps reflexes Acrylamide-exposed 71 71 68 59 4/7 54 44 41 41 39 29 29 27 22 20 20 10 10 Controls - - - - - - - • - - - 10 - . - - - 32 ------- TABLE 7 Comparison of vibration thresholds (VU ± SD) between acrylamide workers and referents Acrylamide Workers Reference Group Age (years) n fingers toes n fingers toes <31 32 2.33±1.05* 5.13 ±3.80* 42 1.20 ±0.29 2.40 ±0.85 31-40 9# 2.19 + 0.90* 4.94 ±2.74* 37 1.38 ±0.37 2.85 ±0.85 >41 0 - 26 1.49 + 0.36 3.53 ±0.97 * By variance analysis and Q test, p < 0.01. # One worker aged 42 was included 33 ------- Electroneuromyographic measurements The mean duration and amplitude of motor units and the percentage of polyphasic potentials in muscles abductor pollicis brevis, abductor digiti minimi and anterior tibialis of the acrylamide group were within normal range. No spontaneous denervation potentials were found during mild contraction of sampled muscles. However, 20 out of 94 sampled muscles showed mixed patterns of recruitment during maximal contraction; 9 samples were of discrete pattern. Abnormal recruitment patterns were also seen in 75% of the 12 samples of anterior tibialis muscles of acrylamide workers. In comparison with the reference values of ENMG parameters on a group basis, there was a prolongation of distal motor latencies in median, ulnar and peroneal nerves as well as a slowing of MCV of peroneal nerve in the acrylamide group. A prolongation of sensory distal latencies and a decrease of sensory potentials were also found in the median, ulnar and sural nerves, as well as a slowing of SCV of sural nerve in acrylamide workers (Table 8). The neurotoxicity index as predictor of clinical diagnosis of peripheral neuropathy The neurotoxicity index (Table 2) in the workers with a clinical diagnosis of peripheral neuropathy and those without are shown in Figure 7. As evident from the plots there is overlap only for two individuals between the neurotoxicity index and the clinical diagnosis, which seems to indicate that the neurotoxicity index as defined is a useful quantitative indicator of acrylamide-induced peripheral neuropathy. In the studied group no worker had a Neurotoxicity Index exceeding 30 points out of the maximal value of 50. Hb adduct levels in humans exposed to acrylamide The Hb adduct levels determined in the workers in the factory in Xinxiang are presented in Table 9. The AA and AN valine adducts were determined using the modified Edman degradation procedure. The GA valine adducts were determined after total hydrolysis of the globin. Since this method is very time-consuming, only 6 such samples were analyzed. Typical mass fragmentograms from the analyses are shown in Fig. 8a and b. All exposed workers had detectable Hb adduct levels of AA (0.3 - 34 nmol/g Hb) and AN (0.02 - 66 nmol/g Hb). GA adducts were detected in the samples from the 5 exposed persons (1.6 - 32 nmol/g Hb) but not in the control, and there was an increase in GA adducts with increasing AA adduct level with a linear regression correlation coefficient of r = 0.96 (Fig. 9). In controls, AA adducts were detected in 1 out of 10 samples (0.01 nmol/g Hb), and AN adducts were detected only in the smokers (in 4 out of 5 smokers, 0.03 - 0.14 nmol/g Hb). Table 10 shows the average Hb adduct levels of A A in the workers according to their location in the factory together with the geometric means of the AA air concentrations. The Hb adducts of AN were also determined on coded samples in another laboratory (T. Fennell, C.I.I.T.). All results are shown in Table 11. In this parallel analysis ANVal adducts were detected also in controls, albeit at low levels. 34 ------- TABLE 8 Comparison of Motor (MCV) and Sensory (SCV) Nerve Conduction between the Acrylamide and Reference groups (mean ± SD). ENMG Parameters Acrylamide group Reference group (N= 41) (N= 80) MCV (m/sec) Median nerve 56.8 + 7.2 59.6 + 5.0 Ulnar nerve 60.0 + 9.2 62.5 + 5.4 Peroneal nerve 43.8 + 8.5* 48.1 + 3.5 Motor Distal latency (ms) Median nerve 4.6+1.3* 3.5 + 0.4 Ulnar nerve 3.1 + 1.1* 2.3 + 0.3 Peroneal nerve 6.0 + 1.9* 3.9 + 0.5 SCV (m/sec) Median nerve 61.6 + 5.8 60.3 + 4.1 Ulnar nerve 61.9 + 6.4 66.9 + 4.3 Sural nerve 46.6 + 6.6* 56.8 + 4.0 Sensory Distal Latency (ms) Median nerve 2.8 + 0.4* 1.8 + 0.3 Ulnar nerve 2.5 + 0.8* 1.6 + 0.2 Sural nerve 3.0 + 0.8* 2.4 + 0.2 *p <0.02 by Student's t-test 35 ------- 35 -r 30 4- 25 + 20 4- X "x 5 15 -f 10 -f 5 4- • • • 8 0 No clinical diagnosis Clinical diagnosis " Figure 7. Neurotoxicity Index in workers with and without clinical diagnosis of peripheral neuropathy. 36 ------- TABLE 9 Hemoglobin adducts of acrylamide (AAVal), glycidamide (GAVal) and acrylonitrile (ANVal) in workers Subject No. Exposure Smoker Hemoglobin adducts (nmol/g Hb)c Job position Cl C2 C3 C4 C5 C6 C7 C8 C9 CIO El E2 E3 E4 E5 E6 E7 E8 E9 E10 Ell E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 E27 E28 E29 E30 E31 Control no no no yes no yes yes yes no yes Exposed ti it it it ti tt ii ti n n n "* it ii it i i t i t • it ii ii " it it it it n " ti AAVal nda nd nd 0.01 nd nd nd nd nd nd 6.9 6.7 1.6 4.8 2.6 24 12 11 0.6 8.5 34 12 5.6 6.7 7.6 5.8 0.3 11 7.6 4.2 11 4.5 3.2 14 3.7 10 8.5 21 5.4 1.3 4.9 GAVal ANVal nd nd -* nd nd 0.14 nd nd 0.03 0.06 nd 0.09 17 22 9.1 37 4.3 16 8.3 5.9 9.3 0.12 16 29 66 18 6.4 5.9 14 32 24 2.7 46 26 28 1.6 18 15 8.9 7.3 - 7.2 58 31 14 12 24 2.5 Ambulatory Synthesis Ambulatory Polymerization Polymerization Ambulatory Polymerization Synthesis Packaging Synthesis Synthesis Synthesis Ambulatory Polymerization Synthesis Polymerization Synthesis Polymerization Polymerization Polymerization Ambulatory Packaging Packaging Polymerization Packaging Ambulatory Synthesis Synthesis Ambulatory No AA for 4 months Polymerization 37 ------- Table 9 continued E32 E33 E34 E35 E36 E37 E38 E39 E40 E41 28 7.1 6.7 3.8 12 7.5 21 32 32 0.6 12 36 0.02 5.7 7.9 21 15 20 28 1.4 22 Synthesis Polymerization Polymerization Synthesis Synthesis Packaging Synthesis Synthesis Store house Ambulatory a nd = Not detected b Not analyzed c The Hb adducts of AA and AN were determined with the modified Edman degradation method, and the adducts of GA were determined in hydrolyzed Hb samples. 38 ------- 1S9C ZJ63338. ANVal IW2 I 1166. 352 375 j \ d4-HOEtVal 54,636. (see 1580 i6oe 1U24 |1i33 11:42 11:51 l\ T 1881 IC4K30. AAVal tese tm 13:36 I3:« U28 SCAN 14:82 €22 (26 M36 S:?5 1153 GAVal T dg-GAVal ; v_ V36 1150 9:35 nee 9:*« 1176 5:45 nee 75056. 125056. 1)96 9-. 55 wee SCAN 16; ee ii* Figure 8. Mass fragmentogram of A A and GA valine adducts. 39 ------- 35 30 - E C 15 g 10 10 15 20 25 30 AAVal (nmol/g Hb) 35 Figure 9. Correlation between AA and GA Hb adducts in workers exposed to AA. 40 ------- TABLE 10 Air concentrations of AA and AAVal Hb adduct levels in workers in different locations Location AAVal* (nmol/g Hb) AA air concentrations* (mg/mj) AAVal expected from air concentrations0 (nmol/g Hb) Controls Packaging Polymerization Ambulatory Synthesis 0.0 ±0.0 (10) 3.9 ± 2.5 (5) 7.3 ±3.4 (12) 9.6 ±6. (8) 14.7 ±10.6 (14) 1.52(0.19-8.8,12) 0.93 0.73(0.11-3.01,18) 0.44 a Mean ± SD (n) b Geometric mean (range, n) c Cumulative in vivo dose over 17 weeks calculated as: 1.52 mg (m3)-1 x (0.2 liter min-' kg bw1) x (60 min hr1) x (8 hr day1) x (6 day week-') x 17 weeks = 1000 liter (m3)-1 x (71 mg mmoH) x 0.5 hr1 = 0.419mMhr Expected AAVal adduct level calculated as (cf. Equ. 1-2): 0.419 mMhr x 4.4 x 10'6 liter (g Hb)-1 hr1 x 1/2 = 0.93 nmol/g Hb 41 ------- TABLE 11 Individual values for parameters predictive of adverse health outcome in workers exposed to acrylamide nr freeAA# merc## AAVal### ANVal* handvibr** footvibr** job employment* NIn cl c2 c3 c4 c5 c6 c7 c8 c9 clO 9 622 23 25 37 4 5 7 14 16 18 19 20 24 26 33 34 1 3 6 13 21 nd nd. nd 65 69 62 nd nd nd nd nd nd nd 251 67 nd nd nd nd nd nd nd nd nd 112 nd 78 nd nd 200 85 nd nd 2 nd 1.6 2.4 2 3 2.4 3 2.4 9 33 47 258 46 27 19 15 19 15 17 35 16 • 78 133 21 27 9 12 12 40 52 0 0 0 0.01 0 0 0 0 0 0 0.6 4.5 3.2 3.7 7.5 4.8 2.6 12 6.7 5.8 11 7.6 4.2 14 10 7.1 6.7 6.9 1.6 24 5.6 11 , 0.09 0.15 0.03 0.33 0.035 0.17 0.25 0.3 0.05 0.41 18.5 22.2 19.5 10.6 16.2 33.2 8.6 8.7 21.6 27.5 7.8 39.6 31.2 10.1 28.9 0.36 9.6 18.2 15.2 11.9 13 21 1.2 1.6 2.1 2 1.6 1.6 1.3 1.8 1.1 1.3 2.6 1.3 2.3 1.6 4.7 3 1.6 2.3 1.7 2.6 1.1 3.1 1.4 1.2 4.3 1.6 1.8 2.8 1.2 1.7 2.6 2.3 3.3 2.8 3 1.5 3.3 2.3 2.1 4.3 4.2 3 4.2 3.1 7.5 2.6 10.2 5.7 2.9 5.2 4.5 4.3 2.4 6.2 3.3 1.4 10.5 2.4 2.5 7 2.2 3.1 6 5.9 control control control control control control control control control control packaging packaging packaging packaging packaging polymerization polymerization polymerization polymerization polymerization polymerization polymerization polymerization polymerization polymerization polymerization polymerization ambulatory ambulatory ambulatory ambulatory ambulatory 0 0 0 0 0 0 0 0 0 0 1.9 1.75 1.4 2.3 2.5 2.5 0.75 1.9 2.4 7 1.1 0.6 3.2 1.4 8.2 6 8.4 8 3.1 1.5 2.75 3.2 0 0 0 0 0 0 0 0 0 0 4 8 8.5 0 24 9.5 9 12 7.5 7.5 2 20 10 4 22 8 9 8.5 4.5 2 8.5 12.5 (continued) ------- TABLE 11 (Continued) Individual values for parameters predictive of adverse health outcome in workers exposed to acrylamide nr freeAA# merc## AAVal### ANVal* haridvibr** footvibr** job employment* NIn 29 41 2 8 10 11 12 15 17 27 28 32 35 36 38 31 39 30 40 nd nd nd 141 109 130 93 nd 65 nd 155 45 251 140 180 nd nd 43 nd 42 129 12 42 78 39 47 23 9 94 28 109 36 163 104 28 318 nd 36 5.4 12 6.7 11 8.5 34 12 7.6 0.3 8.5 21 28 3.8 12 21 4.9 32 1.3 0.6 : 13.5 20.1 14.4 0.22 23.9 26.9 17.9 19.7 22.2 25.9 23.4 26.8 10.6 22.4 26 4.6 32.7 23.7 2.1 1.9 4.3 1.6 1.4 2.6 5.6 2.9 2.1 2.5 3.6 4.7 4.2 1.3 2.3 2.7 1.2 1.5 2 1.5 6.5 4.4 3.1 27 5.8 15.2 7.5 3.3 5.7 7.5 11.9 18.9 2.2 5.7 4.4 2.2 1.9 2.2 2.7 ambulatory ambulatory synthesis synthesis synthesis . synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis synthesis ++ ++ +++ +++ 11.5 4.5 3.5 8 3.75 3.1 3.1 1.6 0.8 5.4 7.9 7.7 2.5 7.5 3 0.1 0.25 1.2 3.5 11 32 16.5 1.5 26.5 34 26 15.5 5 25.5 24.5 26.5 2.5 17 28 2 6.5 12 11.5 P ppm in plasma. ¥ mercapturic acids, jimol/L urine. ¥. nmol/g Hb (from Bergmark et al. 1993). nmol/g Hb (from McNeela et al., in preparation). Vibration units. * years. ** Less than 6 months exposure to AA. *** not exposed to AA during the 4 last months. nd = not detected. ------- Calculation of in vivo doses f Under the assumption that the adducts are stable during the lifetime of the erythrocytes, the daily increment of adducts in a chronic exposure situation, ([RY]/[Y])d, can be calculated as: ([RY]/[Y])tt = ([RY]/[Y])d x iJ2 (1) where ([RY]/[Y])ttis the steady-state level of adducts at the nucleophilic center, Y, and ^ is the life-span of the erythrocytes, approximately 120 days in man (Osterman-Golkar et al., 1976). The daily in vivo dose, D, can then be calculated from: D = ([RY]/[Y])4 / ky (2) where ky is the second-order rate constant for the reaction of the electrophile with the nucleophilic center Y in Hb (in this case Y = -NH2 of the N-terminal valine). As an example, in worker No. E39, the daily in vivo dose of AA is given by: D= 32 nmol/g Hb x 2 = 0.12 mMhr/day (3) 120 days x 4.4 x 10"6 liter (g Hb x hr)'1 and in analogy, the daily in vivo dose of GA is calculated as 0.048 mMhr in the same person. Other biomarkers of exposure The individual data for the concentrations of free acrylamide in plasma (determined by K. Crofton, USEPA, HERL, RTF), S-(2-carboxyethyl) cysteine in urine and Hb adducts of acrylamide and acrylonitrile are shown in Table 11 together with the job classifications of the different workers. For a majority of workers the levels of free acrylamide in the plasma could not be measured above the level of detectability. For the other biomarkers of exposure, statistically significant differences were observed between the exposed and control groups, which shows that they are in principle useful as biomarkers for workers heavily exposed to acrylamide and/or acrylonitrile. The group means for the different categories of workers are shown in Table 12. Biomarkers of exposure as predictors of neurotoxicity The relationships between free acrylamide in the plasma, mercapturic acids in the urine, hemoglobin adducts of acrylamide or acrylonitrile and the lifetime in vivo doses of acrylamide with the neurotoxicity index are shown in Figures 10-14. The correlation coefficients and statistical significances of these relationships are shown in Table 13, assuming linear relationships between the independent and dependent variables. The mean 44 ------- Table 12 Biomarkers according to job classification controls Free AA* mere ac.** AAVal# ANVal# handVU## footVU## 65 ±4 packaging 159 polymeriz 95 ambulatory 143 synthesis 131 ± 59 1.9 ±1.1 0.0 ±0.0 0.18 ±0.13 1.6 ±0.3 3.0 ±0.9 78.6 ±102 3.9 ±2.5 17.4 ± 4.4 2.5 ±1.3 5.5 ± 3.2 32.7 ±36.4 7.7 ±3.4 18.9 ± 12.8 2.1 ± 1.0 4.3 ± 2.5 36.6 ±44.9 9.5 ±7.3 16.1±3.6 2.4±1.0 5.0±1.8 60.3 ± 45.8 13.4 ± 9.8 20.2 ± 7.7 2.9 ± 1.3 7.2 ± 5.1 Life-time AA§ NIn 0.0 ± 0.0 0.0 ± 0.0 8.1 ±6.6 8.9 ±9.1 27.0 ±23.9 10.0 ±5.8 37.6 ±21.9 11.3 ±9.8 68.3 ± 64.2 19.2 ± 10.6 * Means based only on the values above detectability, ppm ** Mercapturic acids, pmol S-(2 carboxyethyl)cysteine/L hydrolyzed urine #nmol/ g Hb ## Vibration units § Life-time in vivo doses of acrylamide; mMhr ------- X c X o c z 200 Concentration of free acrylamlde In plasma (ppm) 300 Figure 10. Correlation between neurotoxicity index and levels of free AA in plasma. 46 ------- 0 100 200 300 Concentration of S-(2-carboxyethyl)cystelne In urine (umol/24 hrs) Figure 11. Correlation between Neurotoxicity Index and urinary mercapturic acid levels. 47 ------- X « T3 X o g « 0 10 20 30 40 Acrylamldd edducts to N-termlnal vallne In hemoglobin (nmol/g Kb) Figure 12. Correlation between neurotoxicity index and AA Val adducts. 48 ------- 0 10 20 30 40 Acrylonltrllo adducts to N-termlnal vellne In Hb (nmol(g Hb)-1) Figure 13. Correlation between neurotoxicity index and AN Val adducts. 49 ------- 40 i 100 200 Accumulated In vivo doses of acrylamlde (mMhr) 300 Figure 14. Correlation between neurotoxicity index and lifetime in vivo doses of AA. 50 ------- TABLE 13 Correlation coefficients and levels of statistical significance for the relationships between different predictive parameters and the Neurotoxicity Index (NIn). X variable Y variable Corr. Coeff. P-value free AA mere AAVal AAVal x time ANVal ANVal x time time Foot Vibratron Hand Vibratron AAVal NIn NIn NIn NIn NIn NIn NIn NIn NIn ANVal 0.15 0.42 0.67 0.60 0.69 0.60 0.44 0.69 0.81 0.44 0.31 <0.01 <0.001 <0.001 <0.001 <0.001 <0.01 <0.001 <0.001 <0.01 51 ------- values of the different biomarkers, as well as of different indicators of neurotoxicity in the different job categories, are shown in Table 12. General discussion of Aim 2 Hemoglobin adducts Electrophilic compounds react with nucleophilic amino acid residues in Hb, such as the SH of cysteine or the NH2 of N-terminal valine. The activated double bonds of AA and AN react with nucleophilic sites in Michael type additions, whereas the epoxide GA reacts according to a classical nucleophilic substitution mechanism. When analyzing samples from humans exposed to both AA and AN, the modified Edman degradation was our method of choice. The reasons for this were two-fold: first, this method is less time-consuming than the determination of adducts following total hydrolysis and ion-exchange chromatography; second, the total hydrolysis method does not distinguish between adducts originating from AA and AN, since both the 2-carbamoylethyl- and the 2-cyanoethyl- groups are converted to the 2-carboxyethyl- group in strong acid. In our analyses, the amount of the reagent PFPITC was increased compared to the standard procedure of Tornqvist et al. (1988) because this has been shown to give a better yield of certain PFPTH-derivatives, and was therefore assumed to increase the yield of AAVal-PFPTH. d4-HOEtVal was used as an internal standard because of the availability of a well-characterized standard globin alkylated with d4-ethylene oxide. The overall response (a combination of yield in the derivatization and extraction procedure, and the response on GC/MS) was for AAVal-PFPTH somewhat lower (1.5-8 times) and for ANVal-PFPTH slightly higher (2-6 times) than for d4-HOEtVal-PFPTH. The yield and extraction efficiency were not determined, but they were assumed to be constant for AAVal-PFPTH and ANVal-PFPTH versus the internal standard. The difference in response using GC/MS was compensated for by the use of calibration curve. Although the PFPTH derivative of GAVal was also formed and was detectable, the overall response was low (1-2 orders of magnitude lower than that of d4-HOEtVal-PFPTH), and the quantitations were not reproducible. This might be explained by the fact that the GA adduct is very hydrophilic so that its PFPTH-derivative may be difficult to extract from the reaction mixture. The AA and GA valine adducts were generally not detected in unexposed controls and a possible background could not be established. AN adducts were, however, detected in most smokers. Based on the lowest levels detected, detection limits for the method appear to be 0.01 nmol/g Hb (signal-to-noise ratio 6:1) and 0.02 nmol/g Hb (signal-to-noise ratio 12:1) for the AAVal-PFPTH and ANVal-PFPTH derivatives, respectively. These results show that the determination of AA and AN adducts using the modified Edman degradation method can be used as a tool to monitor total exposure to AA and/or AN. As discussed above, adduct formation by GA to N-terminal valine was analyzed using the 52 ------- total hydrolysis method, because of the low yields and hydrophilicity of its PFPTH-derivative. The detection limit of this method was about 1 nmol/g Hb. This acid hydrolysis method was used also to determine adduct levels in Hb treated in vitro with AA or GA for the determination of rate constants with cysteine-SH and valine NH2. Background levels of the cysteine adducts were high in unexposed controls, probably due to methodological artifacts, and analysis of these adducts is suitable only for determination of high alkylation levels such as those in the in vitro experiment. The valine adducts (CEVal and CHEVal) did not give the expected structures following derivatization with methanol/HCl and HFBA, apparently due to rearrangement reactions. The resulting derivatives are interpreted as being formed during the acylation, presumably by nucleophilic attack of the N-acyl-oxygen on the carbonyl of the "C-terminal ester" followed by elimination of CH3OH. The derivatives of the dg-analogues therefore contain only six of the original deuteriums in the dg-valine as was shown by the fragmentation pattern on GC/MS. Comparison of mass spectra of the deuterated and the non-deuterated derivatives in the El and the PCI modes proved to be essential in order to elucidate these structures. The CHEVal derivative gave four additional peaks on GC, whose structures were identified as stereoisomers of lactons, also formed during the derivatization. The use of deuterated standards compensated for variations in yield of the different derivatives. The quantitation of GA adducts was made on the same type of derivative as the one formed for CEVal. While performing the analysis of GA valine adducts using total hydrolysis, CEVal was also determined and found to correspond well (± 8 %) to the sum of the AA and AN valine adducts determined by the modified Edman procedure in the same individuals. As mentioned earlier, both AAVal and ANVal form CEVal upon acid hydrolysis of globin. Based on their roles and positions in the production process we have subdivided the AA factory workers into four different categories: (1) workers involved in the synthesis of AA from AN and in the transfer of 35% AA solution into barrels, (2). ambulatory personnel such as foremen, maintenance workers, etc., with no stationary work place, (3) workers involved in the copolymerization of AA and acrylic acid, and (4) workers who packaged the copolymer after mixing it with starch. Among the workers with the lowest levels of AAVal adducts, one had been free of AA exposure for the past four months (No. 30) and one worked in the store house (No. 40). The air levels at different locations in the workshop were monitored on four different occasions prior to blood sampling. In September 1991 the AA air levels ranged between 0.1 and 1.6 mg/m3, and in the summer months the levels ranged between 0.3 and 8.8 mg/m3. Although the concentrations measured fall within a quite narrow range, an occasional level of 153 mg/m3 was recorded during discharge of AA. The maximal allowable air concentration for AA in the workplace is 0.3 mg/m3 both in the People's Republic of China and in the United States. A few conclusions may be drawn from the data presented. First, the average Hb adduct level of AA is related to the job description and to the location of the different workers in the factory. Second, it appears that only a small fraction of the total amount of AA absorbed by the workers 53 ------- originated from inhalation of AA. Indeed, assuming a breathing rate of 0.2 1 min'1 kg'1, estimated for workers doing work involving moderate physical exercise (Calleman et al., 1978), and that the first-order rate of elimination of AA in humans is the same as in rats (0.5 hr1), it may be estimated that the observed Hb adduct levels in workers in the synthesis and polymerization rooms are 10-30 times higher than would be expected from the measured concentration of AA in the air. Our Hb adduct data are thus consistent with the notion that dermal exposure may be the predominant route of uptake, as suggested in several previous studies (EPA, 1988). In addition, the observation of skin peeling of the hands in several of the workers also suggests that dermal exposure is a significant route of uptake of AA. The ratio of the in vivo doses of GA and AA was on average 3:10. In the rat, the percentage of AA converted to GA is about 60% following a low dose of AA, and the conversion of AA to GA decreases as the injected amount of AA increases (Bergmark et al. 1991). The metabolism of AA in the rat is therefore saturated at high administered amounts. It might be assumed that no saturation of metabolism of AA to GA occurs at the exposure levels found in this study in humans, because of the linear correlation between AA and GA adducts. Recent preliminary experiments by D. Segerback (personal communication) showed that in mice and rats treated with AA, the major DNA adduct is N-7-(2-carbamoyl-2- hydroxyethyl)-guanine, which is formed by the reaction of GA with DNA. This is consistent with the hypothesis that GA may be the agent primarily responsible for cancer initiation following exposure to AA (Calleman et al. 1990). The findings that AA is metabolized to the genotoxic metabolite GA in humans, and that GA adducts are linearly related to AA adduct levels, suggest that exposure to AA might be associated with an increased cancer risk. However, a recent epidemiological study (Collins et al. 1989) has not found an association between AA exposure and an excess of cancer deaths, although the exposure levels determined in that study were much lower than in the present one. In this regard, it is of interest to note that the daily in vivo doses of AA and GA in the most highly exposed workers correspond to the in vivo doses in rats injected daily with 3 mg/kg AA. In cancer tests (Johnson et al., 1986), a daily dose of 2 mg/kg increased several types of tumors in Fisher 344 rats. In addition, exposure to AN is also expected to pose an increased risk of cancer. Therefore, follow-up studies using Hb adducts as biomarkers for monitoring reductions in the exposure to AA and AN as well as prospective studies of cancer incidences in workers heavily exposed to A A and AN are warranted. field study As discussed before, the combination of uptake of acrylamide through different routes is a common characteristic of industrial exposure to acrylamide and speaks in favor of using biomarkers reflective of the total uptake of the substance through all routes as a basis for evaluating hazardous conditions in the workplace. 54 ------- The biomarkers of exposure measured in this study reflect, however, different time-periods of exposure and are thus not expected to be equally useful for predicting an effect which is believed to be a function of a cumulative dose, or integrated concentration of the neurotoxic agent. The concentration of free acrylamide in the plasma could only be detected in a few workers from each group except for the most extensively exposed group located in the synthesis room. It must be assumed that the levels of acrylamide detected in three of the controls reflect an artifact peak eluting at the retention time of the derivatized acrylamide, since no Hb adducts could be found from acrylamide in this group, and the levels detected (62-69 ppm) are only slightly above the detectability of this method (40 ppm). Given that this indicator reflects a momentary level and that blood sampling was performed at different times after the workers had left their work positions, the fact that free acrylamide could not be found in a high number of workers and that it correlated poorly with the Neurotoxicity Index, is not surprising. It is, however, noteworthy that the levels of free acrylamide in the plasma of the most highly exposed group are close to what would be expected from the Hb adduct values under the assumption of a rate of elimination of acrylamide from human plasma similar to that estimated in rats (0.5 hr1, Miller et al., 1982; Bergmark et al., 1991). Thus, the average level of acrylamide Hb adducts (14.7 nmol/g Hb) in this group corresponded to an average in vivo dose of 6.6 mMhr during the life-span of the erythrocytes (17 weeks in humans). This corresponds to a steady-state level of 580 ppm of free acrylamide in the plasma of workers working 8 hrs/day, 6 days/week. While quantitative inferences from these types of measurements should be made with great caution, the results seem to indicate that the rate of elimination of acrylamide is of the same order of magnitude in humans as in rats. In contrast, the determination of mercapturic acids reflects exposure during approximately 1 to 2 days prior to urine sampling. In our study, the urine was hydrolyzed prior to analysis to yield the amino acid S-(2-carboxyethyl)-cysteine, the concentration of which reflects not only acrylamide, but also acrylonitrile conjugates, which are hydrolyzed to the same amino acid. In addition to this drawback, it should be kept in mind that mercapturic acids are detoxification products which are not necessarily proportional to the amount of reaction with target enzymes in nervous tissue. To the extent that interindividual variability in detoxification efficiency of acrylamide or acrylonitrile comes into play, the amount of mercapturic acids excreted in the urine may in fact be inversely related to the in vivo doses. Not surprisingly, the concentration of these mercapturic acids in 24-hr urine is a relatively poor predictor of the Neurotoxicity Index. Hemoglobin adducts reflect the in vivo dose (integrated concentration) of an electrophilic substance during the 4-month period prior to blood sampling. As expected, this biomarker is better correlated with the neurotoxicity index than the other biomarkers of exposure determined in this study. Two peculiarities deserve attention in this regard: first, the fact that the Hb adducts formed by acrylonitrile correlate better with the Neurotoxicity Index than those formed by acrylamide, and second, that the lifetime in vivo doses of either agent 55 ------- as estimated from the Hb adduct levels and time of employment do not correlate as well with neurotoxicity as do the Hb adducts themselves. The good correlation between Hb adducts of acrylonitrile and the neurotoxicity index cannot be explained by a causative relationship since acrylonitrile exposure does not result in the different signs, symptoms and predictive parameters which were included in this index as defined here. Humans who have been exclusively exposed to acrylonitrile in occupational settings may present effects on the central nervous system but not the peripheral neuropathy characteristic of acrylamide exposure (F. He, personal communication to C. Calleman). However, although Hb adducts of acrylonitrile correlate better than those of acrylamide in univariate regression analysis, multivariate analysis gives the relationship: NIn = 0.59 (+0.14) x AAVal + 0.43 (+0.08) x ANVal, where both AAVal and ANVal are correlated with the NIn at the p < 0.01. The impairment of vibration sensation has been found to be an early sign of acrylamide neuropathy and proved to be related to the vulnerability of Pacinian corpuscles to acrylamide (LeQuesne, 1980). In this study, the prevalence of deficits of vibration sensation in acrylamide workers examined by a tuning fork (C 128) was 41%, whereas 58.5% of the same acrylamide group detected by a Vibratron n vibration sensitivity tester showed an increase of vibration thresholds which were higher than the upper limits of vibration thresholds of the reference group. This suggests that the Vibratron H vibration sensitivity tester is superior to tuning forks in terms of sensitivity, reliability and quantitation for the assessment of impairments of vibration sensation. It is therefore potentially useful in field screening of peripheral nerve dysfunction in acrylamide exposed workers. In fact, vibration thresholds in hands as determined by the Vibratron instrument show the best correlation with neurotoxicity of all the potentially predictive parameters. This good correlation is hardly unexpected given that loss of vibration sensitivity is a more sensitive indicator of peripheral neuropathy than either loss of pain or touch sensitivity and the Vibratron instrument may thus be a comparatively inexpensive and useful tool for identifying workshops with unacceptable exposure conditions. The ENMG findings in acrylamide workers manifested as a prolongation of distal motor and sensory latencies and a decrease of distal sensory potentials in peripheral nerves indicate a damage to the distal part of the peripheral nervous system which coincides with the pattern of the central-peripheral distal axonopathy found in cats and rats dosed with acrylamide. The presence of abnormal recruitment patterns without any spontaneous denervation potentials of the sampled muscles suggests a mild damage to the motor fibers of the peripheral nervous system. On the other hand, there was a significant decrease of distal sensory potentials in 17 out of 41 acrylamide workers, the lowest being undetectable, indicating more involvement of sensory fibers than motor fibers in acrylamide neuropathy (Sumner and Asbury, 1974). According to the diagnostic criteria for occupational acrylamide poisoning proposed in our previous studies (He et al., 1989) and based on the clinical and ENMG data available, 13 56 ------- out of the acrylamide group under study were diagnosed with mild occupational acrylamide poisoning at a prevalence of 31.7%. The results of this study provide evidence of neurotoxic effects of acrylamide in exposed workers, similar to those reported in previous studies (He et al., 1989, LeQuesne, 1980). The symptoms and signs presented in the acrylamide workers include numbness and weakness of hands and feet, excessive sweating of hands, impairments of vibration, pain and touch sensations, as well as diminution and loss of ankle reflexes. These clinical manifestations, usually preceded by peeling of skin from hands, are more prevalent than in the reference group and strongly indicate an involvement of the peripheral nervous system. The results of medical surveillance clearly indicate the necessity of prevention of acrylamide poisoning in the factory under study. The neurotoxicity index was designed to be: (1) specific for acrylamide-induced peripheral neuropathy and to exclude such effects that might be due to acrylonitrile-induced effects on the central nervous system, (2) objective, i.e. independent of the subjective experience of the workers, and (3) reflective of the severity of the neurological disturbance. There was only minimal overlap in the Neurotoxicity Index between those individuals with a clinical diagnosis of peripheral neuropathy and those without. Among the workers there was a better correlation between this neurotoxicity index and the levels of Hb adducts (r = 0.67) than with either the cumulative in vivo dose (r = 0.60) or the duration of employment (r = 0.44). However, in workers employed for more than 2 years, the correlation between neurotoxicity and the duration of exposure virtually disappeared (r = -0.06). The interpretation was thus made that the average dose-rate (as reflected by the Hb adduct levels for the 4-month period prior to blood sampling), rather than the cumulative in vivo dose, is the better predictive parameter for peripheral neuropathy in workers exposed for more than 2 years. A risk analysis was hence performed which included only those workers who had been exposed to acrylamide for more than 2 years. If the percentages of these workers with different diagnostic indicators of neurotoxicity were plotted against their Hb adduct levels, sigmoid curves were evident reaching 100% in the group with the highest level of Hb adducts. From the plot, NOEL and LOEL and EL^ for clinical diagnosis of peripheral neuropathy were estimated to 2, 6 and 7.9 nmol AAVal (g Hb)'1, respectively. Assuming a rate of elimination of acrylamide in man similar to that estimated in the rat, 0.5 hr1, these Hb adduct levels correspond to intakes of 0.3, 0.8 and 1.1 mg/kg/day, and further assuming a 100% uptake of airborne acrylamide and a rate of alveolar ventilation of 0.2 1 min'1 (kg bw)'1 in exposed workers, NOEL, LOEL and ELJO for peripheral- neuropathy correspond to concentrations of 3, 8, and 10 mg/m3, respectively. It may thus be concluded that in workers exposed for more than 2 years, the currently applied TWA of acrylamide, 0.3 mg/m3, has a safety factor of 10 for adverse neurological effects. It should be also noted, however, that in the present study the primary route of exposure to AA appeared to be dermal. 57 ------- AIM 3 Comparative neurotoxicity of acrylamide and glycidamide Rats injected with the lowest dose of acrylamide gained weight, while those administered 50 mg/kg acrylamide or glycidamide did not (Fig. 15). Rats in the 100 mg/kg glycidamide group lost weight during the treatment. Animals in the two high-dose groups started to appear lethargic and ataxic after 5 injections (cumulative doses of 250 and 500 mg/kg for acrylamide and glycidamide, respectively). However, the appearance of the animals was quite different. While acrylamide-treated rats had their hindlimbs extended to both sides in the hindlimb splay test, glycidamide-treated rats routinely extended both legs to the same side. Behavioral tests The results of the rotarod testing are shown in Fig. 16. At the low doses, both acrylamide and glycidamide had no significant effect on the ability of rats to stay on the rod. Similar significant effects were, however, observed starting on day 6 in animals receiving either acrylamide (50 mg/kg) or glycidamide (100 mg/kg). On the other hand, in the hindlimb splay test a significant effect was seen only in animals treated with acrylamide (Fig. 17). At the 50 mg/kg dose, acrylamide increased the spread of the hindlimbs on landing after two doses, while at the 25 mg/kg dose a significant effect was seen after 8 injections. Neither dose level of glycidamide had any effect in this test. In the open field tests, no significant differences were found between control and treated rats at any time point, with regard to deambulation. However, animals in the 50 mg/kg acrylamide group showed a decrease in rearing on day 4. On day 8 the values for rearing (mean score jf SEM) were: control, 26.9 ± 9.4; glycidamide 50 mg/kg, 21.6 ± 9.6; glycidamide 100 mg/kg, 13.0 ± 2.6; acrylamide 25 mg/kg, 19.5 + 5.1; acrylamide 50 mg/kg, 6.1 ± 2.9 (<0.05; n = 10/group). Morphological observations One of the most striking features at necropsy was urinary retention and distended bladders in rats dosed subacutely with acrylamide, as previously reported. The effect of acrylamide was clearly dose-dependent. Glycidamide appeared to cause some urinary retention but to a much lesser degree. In animals treated for 8 days with 50 mg/kg acrylamide or 100 mg/kg glycidamide, no apparent morphological alterations were seen at the light microscopic level in sections of the sciatic nerve. Therefore, additional groups of rats were injected with the same doses for 11 or 12 days to reach cumulative doses of 600 mg/kg (acrylamide) or 1200 mg/kg (glycidamide). Characteristic changes of acrylamide neurotoxicity were seen in the acrylamide group, with degeneration and axonal swelling, but not in the glycidamide-treated animals. 58 ------- 320 250 4 6 Days of treatment Figure 15. Body weights of rats treated with AA or GA. 59 ------- o 0 •o o c o 0 E 0 o CO --•O-- Control AA25 AA50 GA50 GA100 4 6 Days of treatment 10 Figure 16. Rotarod test performance of rats exposed to AA or GA. 60 ------- 4 6 Days of treatment ••D-- -A- Control AA25 AA50 GA50 GA100 10 Figure 17. Hindlimb splay test in rats exposed to A A or GA. 61 ------- Male reproductive toxicity of acrylamide and glycidamide The effects of AA and its epoxide metabolite GA on the male reproductive system were determined after 7 and 14 days of treatment, respectively (50 mg/kg i.p./day). GA was administered for 14 days and no overt signs of toxicity were seen, except for a significant decrease in total body weight. Administration of AA was stopped after 7 days because at this time rats showed severe signs of neurotoxicity, i.e., hindlimb weakness and ataxia. Body weight following AA treatment was also significantly decreased. GA significantly decreased epididymis weight, the ratio of epididymis weight/testis weight and the protein content of testicular tissue (Table 14), while AA exposure had no effect on these parameters. The vas deferens sperm count was significantly decreased by both AA and GA; however, only GA significantly affected viability (control, 33.1 +. 7.7% viable; GA, 3.6 ± 1.4% viable) (Table 15). DSP was affected in the epididymis only by GA, but no effect in the testis was seen for either treatment. Plasma testosterone levels were also unaffected by either treatment. No differences in gross morphology and light microscopy of reproductive organs were seen with either compound. The seminiferous tubules were unaltered in their epithelium. Sertoli cells and interstitial cells appeared to be unaffected with either treatment. General discussion of Aim 3 The main conclusion which can be drawn from the present results is that GA is involved in the reproductive toxicity of AA, but not in its neurotoxicity. Administration of AA, to a cumulative dose of 400 mg/kg, had a significant effect on the performance of rats in the rotarod test and in the hindlimb splay test, which is considered more specific for peripheral neuropathy (Edwards and Parker, 1977). Indeed, A A had a significant effect in this latter test even at the lower dosage regimen (25 mg/kg/da, for a cumulative dose of 200 mg/kg). GA, at the cumulative dose of 400 mg/kg, had no effect on either test, while at the cumulative dose of 800 mg/kg a significant effect was seen in the rotarod test. Animals in this group were very weak and lost weight compared to the other groups; the positive effect observed in the rotarod may, therefore, be due to general systemic toxicity rather than to a neurotoxic effect on peripheral nerves. The lack of effect in the hindlimb splay test seems to confirm this hypothesis. Furthermore, morphological observation at the light microscope level did not identify any damage to the tibial and sciatic nerve of GA 100 - treated rats. Where male rats were given A A in a dosage regimen of 50 mg/kg/day for a cumulative dose of 350 mg/kg, mild signs of reproductive toxicity were present, as shown by a decrease of vas deferens sperm count. Longer treatments with AA were prevented by its severe neurotoxicity. On the other hand, when GA was given at the same dosage regimen for a cumulative dose of 700 mg/kg, signs of toxicity were minimal and no neurotoxicity was observed, although animals displayed significant toxic effects in the reproductive 62 ------- TABLE 14 Effects of acrylamide and glycidamide on male reproductive organs in rats Body wt (g) Testis wt (g) Epididymis wt (g) Testis wt/body wt (%) Epididy wt/testis wt (%) Testis protein (mg/g testis) Testosterone (ng/ml) Control8 372 ±3.7 3.7 + 0.1 1.2 ±0.04 1.0 ±0.02 33.5+1.4 96.6 ±5.2 0.6 + 0.2 Acrylamide 330 + 80«> 3.4 ±0.1 1.1 ±0.03 1.0 ±0.03 33.1 ±1.1 105.8 ±4.8 0.8 ±0.2 Glycidamide 344 ± 5.5b 3.6 ±0.1 1.0±0.04b 1.1 ±0.04 , 29.1 ±0.9" 77.4 + 4. lb 1.1 ±0.4 * Results are mean ± S.E. of at least 4 animals. No significant difference between controls at 7 versus 14 days. b Significantly different from control, p <0.05. 63 ------- TABLE 15 Effects of acrylamide and glycidamide on male reproductive parameters in rats Control3 Acrylamide Glycidamide Vas deferens sperm count (cell no. x 106) Sperm cell viability (% viability) Testicular DSP (cell no. x 106) Epididymal DSP Testosterone (ng/ml) 14.3 ±2.6 33.1±7.7 67.6 ±18.5 365 ± 39.7 0.6 + 0.2 7.8±0.6b 32.4 ± 6.8 57.6 ±10.2 303 + 25.8 0.8 + 0.2 6.2±1.2b 3.6±1.4b 66.2 ±11.0 68.8 ± 10.3b 1.1 ±0.4 a Results are mean ± S.E. of at least 4 animals. No significant difference between controls at 7 versus 14 days. b Significantly different from control, p < 0.05. 64 ------- system. The finding that GA causes reproductive toxicity agrees with the hypothesis of Sega et al. (1990) who postulated a role for this epoxide metabolite in the effects of AA on male gonads. 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