Hemoglobin adducts from acrylonitrile and ethylene oxide in cigarette smokers: effects of glutathione S-transferase T1-null and M1-null genotypes.

Acrylonitrile (ACN) is used to manufacture plastics and fibers. It is carcinogenic in rats and is found in cigarette smoke. Ethylene oxide (EO) is a metabolite of ethylene, also found in cigarette smoke, and is carcinogenic in rodents. Both ACN and EO undergo conjugation with glutathione. The objectives of this study were to examine the relationship between cigarette smoking and hemoglobin adducts derived from ACN and EO and to investigate whether null genotypes for glutathione transferase (GSTM1 and GSTT1) alter the internal dose of these agents. The hemoglobin adducts N-(2-cyanoethyl)valine (CEVal), which is formed from ACN, and N-(2-hydroxyethyl)valine (HEVal), which is formed from EO, and GST genotypes were determined in blood samples obtained from 16 nonsmokers and 32 smokers (one to two packs/day). Smoking information was obtained by questionnaire, and plasma cotinine levels were determined by immunoassay. Glutathione transferase null genotypes (GSTM1 and GSTT1) were determined by PCR. Both CEVal and HEVal levels increased with increased cigarette smoking dose (both self-reported and cotinine-based). CEVal and HEVal levels were also correlated. GSTM1 and GSTT1 genotypes had little effect on CEVal concentrations. GSTM1 null genotypes had no significant impact on HEVal. However, HEVal levels were significantly elevated in GSTT1-null individuals when normalized to smoking status or cotinine levels. The ratio of HEVal:CEVal was also elevated in GSTT1-null smokers (1.50 +/- 0.57 versus 0.88 +/- 0.24; P = 0.0002). The lack of a functional GSTT1 is estimated to increase the internal dose of EO derived from cigarette smoke by 50-70%.

Monitoring exposure to acrylonitrile using adducts with N-terminal valine in hemoglobin.

Human exposure to acrylonitrile (ACN), a carcinogen in rats, may occur in industrial settings, through waste water and tobacco smoke. ACN is an electrophilic compound and binds covalently to nucleophilic sites in macromolecules. Measurements of adducts with hemoglobin could be utilized for improved exposure assessments. In this study, a method for quantification of N-(2-cyanoethyl)valine (CEVal), the product of reaction of ACN with N-terminal valine in hemoglobin has been developed. The method is based on the N-alkyl Edman procedure, which involves derivatization of the globin with pentafluorophenyl isothiocyanate and gas chromatographic-mass spectrometric analysis of the resulting thiohydantoin. An internal standard was prepared by reacting valylglycylglycine with [2H3]ACN, spiked with [14C]ACN to a known sp. act. Levels of CEVal were measured in globin from rats exposed to 3-300 p.p.m. ACN in drinking water for 105 days and from humans (four smokers and four non-smokers). CEVal was detected at all exposure levels in the drinking water study. The relationship between adduct level and water concentration was linear at concentrations of 10 p.p.m. (corresponding to an average daily uptake of c. 0.74 mg ACN/kg body wt during the 65 days prior to sacrifice) and below, with a slope of 37.7 pmol CEVal/g globin/p.p.m. At higher concentrations, adduct levels increased sublinearly, indicating saturation of a metabolic process for elimination of ACN. Comparison of adduct formation with the estimated dose (mg/kg/day) of ACN indicated that at low dose (0-10 p.p.m.) CEVal = 0.508 x ACN dose + 0.048 and at high dose (35-300 p.p.m.) CEVal = 1.142 x ACN dose - 1.098. Globin from the smokers (10-20 cigarettes/day) contained about 90 pmol CEVal/g, whereas the adduct levels in globin from non-smokers were below the detection limit. The analytical sensitivity should be sufficient to allow monitoring of occupationally exposed workers at levels well below the current Occupational Safety and Health Administration standard of 2 p.p.m.

Molecular dosimetry of DNA and hemoglobin adducts in mice and rats exposed to ethylene oxide.

Experiments involving ethylene oxide (ETO) have been used to support the concept of using adducts in hemoglobin as a surrogate for DNA adducts in target tissues. The relationship between repeated exposures to ETO and the formation of N-(2-hydroxyethyl)valine (HEtVal) in hemoglobin and 7-(2-hydroxyethyl)guanine (7-HEG) in DNA was investigated in male rats and mice exposed by inhalation to 0, 3, 10, 33, or 100 ppm ETO for 6 hr/day for 4 weeks, or exposed to 100 ppm (mice) or 300 ppm (rats) for 1, 3, 5, 10, or 20 days (5 days/week). HEtVal was determined by Edman degradation, and 7-HEG was quantitated by HPLC separation and fluorescence detection. HEtVal formation was linear between 3 and 33 ppm ETO and increased in slope above 33 ppm. The dose-response curves for 7-HEG in rat tissues were linear between 10 and 100 ppm ETO and increased in slope above 100 ppm. In contrast, only exposures to 100 ppm ETO resulted in significant accumulation of 7-HEG in mice. Hemoglobin adducts were lost at a greater rate than predicted by normal erythrocyte life span. The loss of 7-HEG from DNA was both species and tissue dependent, with the adduct half-lives ranging from 2.9 to 5.8 days in rat tissues (brain, kidney, liver, lung, spleen, testis) and 1.0 to 2.3 days in all mouse tissues except kidney (t1/2 = 6.9 days). The concentrations of HEtVal were similar in concurrently exposed rats and mice, whereas DNA from rats had at least 2-fold greater concentrations of 7-HEG than DNA from mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular dosimetry of ethylene oxide: formation and persistence of N-(2-hydroxyethyl)valine in hemoglobin following repeated exposures of rats and mice.

The formation of N-(2-hydroxyethyl)valine (HEVal) in hemoglobin was investigated in male F344 rats (10/group) and B6C3F1 mice (20/group) exposed to 0, 3, 10, 33, 100, or 300 (rats only) ppm ethylene oxide (ETO) by inhalation for 6 h/day for 4 weeks (5 days/week) or exposed to 100 (mice) or 300 ppm (rats) ETO for 1 or 3 days, or 1, 2, or 4 weeks (5 days/week). The persistence of HEVal was studied in animals killed up to 10 days after cessation of the 4-week time-course studies. HEVal was determined by a modified Edman degradation and quantitation of the resulting pentafluorophenylthiohydantoin derivative, using gas chromatography-mass spectrometry. The resulting experimental data were compared to simulations derived with a model for the formation and removal of hemoglobin adducts (T.R. Fennell, S.C.J. Sumner, and V.E. Walker, Cancer Epidemiol., Biomarkers & Prev., 1: 213-219, 1992). Repeated exposures of rats and mice for 4 weeks to 300 and 100 ppm ETO, respectively, led to an accumulation of HEVal that was 14 (rats) and 15 (mice) times greater than that found after 1 day of exposure [28 +/- 2 (SE) and 9.4 +/- 0.4 (SE) pmol HEVal/mg globin in rats and mice, respectively]. After cessation of exposures, HEVal was lost faster than predicted by the normal erythrocyte life span alone. An initial phase of rapid decline in HEVal concentrations was consistent with the removal of older, more heavily alkylated populations of RBCs, accompanied by a burst of erythropoiesis. The dose-response curves for HEVal were linear between 3 and 33 ppm ETO, with 3.5 +/- 0.2 and 3.4 +/- 0.3 pmol adduct/mg globin formed in rats and mice, respectively, after 4 weeks of exposure to 3 ppm ETO. Above 33 ppm ETO, the slope of the dose-response curves increased. Comparison of the dose response for HEVal in rats exposed to ETO for 4 weeks to the dose-response for N tau-(2-hydroxyethyl)histidine in rats exposed to the same concentrations of ETO for 2 years (S. Osterman-Golkar et al., Teratog. Carcinog. Mutagen., 3: 395-405, 1983) suggested that exposures to ETO can reduce the life span of erythrocytes in a concentration- and time-dependent manner. Correlation of the experimental data and simulations for the formation and removal of HEVal demonstrated that perturbations in erythropoiesis and RBC life span complicate the estimation of exposures to ETO when estimates are based upon hemoglobin adduct measurements in heavily exposed individuals.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization and quantitation of urinary metabolites of [1,2,3-13C]acrylamide in rats and mice using 13C nuclear magnetic resonance spectroscopy.

Acrylamide, widely used for the production of polymers and as a grouting agent, causes neurotoxic effects in humans and neurotoxic, genotoxic, reproductive, and carcinogenic effects in laboratory animals. In this study, 13C NMR spectroscopy was used to detect metabolites of acrylamide directly in the urine of rats and mice following administration of [1,2,3-13C]acrylamide (50 mg/kg po). Two-dimensional NMR experiments were used to correlate carbon signals for each metabolite in the urine samples and to determine the number of hydrogens attached to each carbon. Metabolite structures were identified from the NMR data together with calculated values of shift for biochemically feasible metabolites and by comparison with standards. The metabolites assigned in rat and mouse urine are N-acetyl-S-(3-amino-3-oxopropyl)cysteine, N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine, N-acetyl-S-(1-carbamoyl-2-hydroxy-ethyl)cysteine, glycidamide, and 2,3-dihydroxypropionamide. These metabolites arise from direct conjugation of acrylamide with glutathione or from oxidation to the epoxide, glycidamide, and further metabolism. Acrylamide was also detected in the urine. Quantitation was carried out by integrating the metabolite carbon signals with respect to that of dioxane added at a known concentration. The major metabolite for both the rat (70% of total metabolites excreted) and the mouse (40%) was formed from direct conjugation of acrylamide with glutathione. The remaining metabolites for the rat (30%) and mouse (60%) are derived from glycidamide. The species differences in extent of metabolism through glycidamide may have important consequences for the toxic and carcinogenic effects of acrylamide.